This paper studies the total flavonoids extraction and activity in vitro of Rose rugosa cv. Plena residues from Shandong province. Single factor experiment and orthogonal experiment are designed to determine the optimal extraction conditions for flavonoids. The antioxidant and antimicrobial activities of total flavonoids in vitro are evaluated by free radical scavenging method and Oxford cup method. Results show that the optimal extraction process for flavonoids are as follows: set the ethanol concentration to 60% and the solid-liquid ratio of 1∶30, then shake the rose residues at 80 ℃ for 1.5 hours, after which run it ultrasonic treatment for 45 minutes at 140 W and 60 ℃. This process gives us the total flavonoids content of 58.53 mg/g. The extracted flavonoids from rose residues demonstrates good antioxidant activity. The scavenging rates of DPPH· radical, ABTS+ radical and hydroxyl radical are 91.58%, 73.71% and 467.91 U/mL respectively. The total flavonoids have good antimicrobial activity on Staphylococcus aureus and Bacillus cereus. The flavonoids could significantly inhibit Staphylococcus aureus. This study would laid a solid foundation for the comprehensive utilization of Rose rugosa cv. Plena residues.