Sand rice (Agriophyllum squarrosum), a pioneering annual plant thriving in deserts and sandy regions throughout the Asian interior, is believed to be a potential food and forage crop due to its significant nutritional and medicinal values. Previous metabolomics analyses have revealed that sand rice contains abundant flavonoid components, which are known for their wide applications in cosmetics, food, and pharmaceuticals. To optimize the use of flavonoids in sand rice, in this study, the response surface methodology (RSM) was selected to determine the optimal ultrasonic-assisted extraction (UAE) criteria for flavonoids extraction from the aerial part of sand rice firstly. Statistical analyses unveiled the optimum parameters for flavonoids extraction from sand rice could be 62% of ethanol concentration, 1:43 solid-toliquid ratio, 160 W for ultrasound power, and 52 degrees C for extraction temperature with extraction time of 12 min. Under this condition, the experiment optimum total flavonoid yield could reach at 15.24 mg/g, which was correspond to the maximum predicted value of RSM with 15.22 mg/g. Subsequently, the antifungal efficacy of these extracts was evaluated against three common plant pathogenic fungi, showing a significant inhibitory effect with the highest rate of inhibition reaching 25.3% at a concentration of 4 mg/mL, underscoring its potential as a natural antimicrobial agent. This study will not only provide a powerful method to extract flavonoids from a desert resource plant, but also pave the way for industrial development and application of the promising desert plants with high nutritional and medicinal values.