To solve the problem regarding the difficulty of the traditional microwave-induced thermoacoustic imaging technology to reconstruct complex biological tissues containing bones completely and clearly, this study proposes a new dual-frequency microwave-induced thermoacoustic imaging technology. That is, two sets of single-frequency microwave-induced thermoacoustic data were obtained by exciting tissues with two different center frequencies’ short pulse width microwave. After energy calibration of the above two sets of data using regional pixel mean and tissue absorption coefficient compensation, the ratio of the bone’s microwave energy absorption coefficients at two different frequencies is used to weigh and subtract the above two sets of calibrated data. Thereafter, dual-frequency microwave-induced thermoacoustic images were finally obtained to reduce the side effects of bone. In this paper, the principle of the dual-frequency microwave-induced thermoacoustic imaging method is first described in detail. Next, the dual-frequency microwave-induced thermoacoustic imaging system is introduced. Finally, imaging experiments of rabbit knee joints and chicken metatarsal bones are conducted, and the imaging results are qualitatively observed and semiquantitatively analyzed. The experimental results show that compared with single-frequency microwave-induced thermoacoustic imaging, the estimated tissue dimensions and signal trends in dual-frequency microwave-induced thermoacoustic imaging are more consistent with the corresponding actual tissue dimensions and theoretical analysis. Dual-frequency microwave-induced thermoacoustic imaging technology can reduce the “negative contrast” effect of the bone so that the affected meniscus, cruciate ligament, and bone marrow cavity can be clearly and completely reconstructed. This method can improve the imaging ability of microwave-induced thermoacoustic imaging technology for complex biological tissues containing bones, thus promoting the progress of related applied research and providing a reference for other imaging methods to alleviate side effects on bones. © 2024 Chinese Academy of Sciences. All rights reserved.