Minimally invasive techniques for in vivo tissue analysis are desired by life science and medical research. Herein, a new ionization interface coupled with ultrasonic sputter desorption (USD) was developed for in vitro and in vivo tissue analysis. Sample molecules were effectively sputtered out when the high-frequency ultrasonic probe touched the tissue. Then, the sputtered molecules were collected and ionized by a custom-made heated quartz tube and finally analyzed by mass spectrometry (MS) online. The sample pretreatment of the USD-MS technique was quite simple and required no other steps except for wetting the tissue surface with ethanol to assist molecular extraction. Experimental results demonstrated that the proposed method was suitable for the analysis of different morphologies of tissues (such as liver, brain, kidney, and lung) and performed well in the analysis of liver tumors and paracancerous tissues. Moreover, as the proposed method caused little damage to the tissues during analysis, rats and mice with orthotopic tumors still survived after the experiments. Overall, the newly developed USD-MS technique was an effective tool for minimally invasive tissue analysis and could be used as a new candidate method for in situ and real-time analysis of biological tissues in vitro and in vivo.