The present study was conducted to evaluate the influence of ultrasound on bioactive compounds and biological activities of blue butterfly pea flower (Clitoria ternatea L.). For this purpose, optimized conditions (temperature 50 °C, time 150 min, solid to liquid ratio 1 g:15 ml, 70% amplitude and 240 W, 20 kHz frequency, 3 s on and 3 s off) of ultrasound (US) and conventional extraction (AGE: Agitation, water bath for 150 min, 50 °C at 150 rmp) were used. The results showed significant (p < 0.05) effect of US and AGE on total phenolics (TPCs), flavonoids (TFCs) and antioxidant activities (DPPH, ABTS, FRAP, reducing activity, Cu²⁺ and H₂O₂) of butterfly pea flower extract (BPFE). The results showed an increased trend in yield, TPCs, TFCs and antioxidant activities of US treated BPFE with comparison to AGE. However, insignificant (p > 0.05) effect of US and AGE over TFlaCs and PACs were observed. Moreover, the results of Fourier-transform infrared spectroscopy (FTIR) showed little changes in spectrum and US does not affect the functional group of bioactive compounds structure. Additionally, extracts (500-2000 µg/ml) protect pBR322 plasmid DNA damage induced by (1 mM H₂O₂ and 1 mM FeSO₄), plasma oxidation (induced by 250 µM CuCl₂) and inhibit erythrocyte hemolysis (induced by 200 mM AAPH, 34.6 to 66.73%). Sonication can be applied successfully for the extraction of bioactive compounds from plant materials with high biological activities.