Ultrasonic irradiation for modified pectin preparation has become a research hotspot, but the influence of ultrasonic frequency is rarely studied. Therefore, in this study, mono frequency (MFU) and simultaneous dual frequency (SDFU) ultrasounds were used to modify citrus pectin (CP), and the effects of different ultrasonic frequency modes on the physicochemical, structural, functional, and biological properties of CP were comparatively investigated. The results showed that ultrasonic irradiation at 40 kHz in the MFU mode led to a larger increment in the galacturonic acid content of CP, but greater reductions in its intrinsic viscosity, molecular weight, and degree of methoxylation than in the SDFU modes. Ultrasonic frequency had no significant influence on the main CP chain, but rather on its neutral sugar side chains. The scanning electron microscopy (SEM) results suggested that CP treated by ultrasound at 40 kHz in the MFU mode exhibited fewer aggregates or looser networks than other ultrasonic treatments. Compared with the original CP, ultrasound treatment at different frequency modes effectively enhanced the thermal stability of CP, but remarkably lowered its shear thinning fluid behavior and liquid-like property. Moreover, among ultrasonically treated CPs, the CP obtained through ultrasound at 40 kHz in MFU mode exhibited the best antioxidant activity and bile acid-binding capacity in vitro. Display omitted •Ultrasound at different frequencies was used to modify citrus pectin (CP).•Mono frequency ultrasound (MFU) at 40 kHz greatly affected CP's structural features.•Larger changes in rheological and thermal properties of CP caused by MFU at 40 kHz.•Ultrasonic-treated CP at 40 kHz showed a better biological activity than the others.