Abstract: Summer high-temperature waterlogging is a significant environmental stress factor affecting alfalfa growth in North China. This study aimed to investigate the physiological and biochemical responses and quality changes of alfalfa (Medicago sativa L.) to summer high-temperature waterlogging by simulating flooding depth and water temperature, providing data support for alfalfa cultivation under high-temperature waterlogging stress and theoretical guidance for reducing economic losses through timely harvesting during waterlogging. A two-factor randomized block design was adopted, with three flooding levels (W1: 0 cm, level with soil surface; W2: 10 cm above soil surface; W3: 20 cm above soil surface) and four flooding water temperatures (25 °C, 30 °C, 35 °C, and 40 °C), resulting in 12 treatment groups (3 levels × 4 temperatures). The control group (CK) was maintained at air temperature (25±2) °C and 70% field capacity. Samples were collected on the 2nd, 4th, and 6th days to measure alfalfa's nutritional quality, metabolic products, root system vitality, reactive oxygen metabolism, antioxidant enzyme activity, and anaerobic respiration enzyme activity. The results showed that: 1) Flooding water temperature, flooding level, and flooding time significantly affected the physiological and biochemical indicators of alfalfa. 2) Flooding water temperature had a more significant impact on alfalfa's physiological and biochemical indicators than flooding level, especially under high-temperature conditions (40 °C), where alfalfa's physiological functions were almost completely impaired, indicating that high temperature is a key environmental factor leading to rapid alfalfa death during summer waterlogging. 3) With increasing flooding depth, water temperature, and flooding time, alfalfa's survival time shortened, and crude protein (CP), ether extract (EE), soluble sugars in the roots (SSR), and root system vitality (RSV) contents significantly decreased, while malondialdehyde (MDA) content in leaves significantly increased. The activities of ascorbate peroxidase (APX) in leaves, alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC) in roots showed an initial increase followed by a decrease. 4) Under mild stress conditions (e.g., 25 °C flooding water temperature, W1 flooding level, and 2 days of flooding time), CP, EE, relative feed value (RFV), SSR, and RSV significantly increased compared to CK, while neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents significantly decreased. Specifically, EE, SSR, and RSV reached their highest levels in the 25 °C + W1 treatment group on the 2nd day, increasing by 4.76%, 11.83%, and 8.98%, respectively; NDF and ADF reached their lowest levels in the 25 °C + W1 treatment group on the 4th day, decreasing by 10.86% and 4.05%, respectively; RFV peaked in the 25 °C + W2 treatment group on the 2nd day, increasing by 5.27%. These findings indicate a transient quality optimization window, providing a critical time node for disaster harvesting. Although previous studies have revealed the effects of temperature or flooding time on alfalfa under single stress conditions, the physiological and biochemical response mechanisms and quality changes under the combined stress of flooding depth, water temperature, and time remain unclear. This study innovatively elucidates the physiological and biochemical response mechanisms and quality changes of alfalfa under the combined stress of flooding depth, water temperature, and time, providing a theoretical framework and data support for breeding waterlogging-resistant alfalfa varieties, decision-making for disaster harvesting, and precision cultivation management in high-temperature waterlogging regions.