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Paeonia ostii is the representative of oil-utilized peony, its roots (PR) and leaves (PL) are discarded as by-products, resulting in a waste of resources. The exploration of extraction process of active ingredients from PR/PL is beneficial to the comprehensive utilization. In this work, the optimum process of Ultrasound-Assisted Extraction (UAEo) of total flavonoids content (TFC) from PR and PL was determined by single factor and response surface methodology. The results showed that UAEo was 80 min, 1:5 g/mL, 250 W, 33.83 mg Rutin/g dw for PR and 60 min, 1:10 g/mL, 250 W, 25.85 mg Rutin/g dw for PL. Then, Homogenization-Assisted Extraction (HAE), Homogenization-Ultrasound-Assisted Extraction (HUAEo) and Ultrasound-Homogenization-Assisted Extraction (UHAEo) were further analyzed. The highest PR(PL)-TFC by HUAEo at homogenization 5 min was 49.58 ± 0.25 mg/g with an increase of 46.6% (33.02 ± 0.04 mg/g with an increase of 27.7%). The highest PR/PL-total phenolic content by the HUAEo reached 77.84 ± 0.52 mg/g dw and 146.62 ± 2.77 mg/g dw for homogenization 3 min. However, there was no significant difference between HUAEo and UHAEo. In conclusion, the TFC increased with the extension of HAE time, and the combined extraction was higher yield than the single extraction.