To evaluate the value of transrectal ultrasound (TRUS) guided biopsy in endoscopy negative biopsy patients with rectal lesions.150 endoscopy negative biopsy result rectal lesions adopted the transrectal ultrasound-guided biopsy. Based on whether the patients received contrast-enhanced ultrasound examination or not before the biopsies, all enrolled cases were divided into TRUS guided group and contrast-enhanced TRUS (CE-TRUS) guided group, and the safety and diagnostic performances were analyzed retrospectively.We obtained adequate specimens in the majority of cases (98.7%,148/150); There were no complications identified in our study. 126 patients received contrast-enhanced TRUS examination before biopsy to evaluate vascular perfusion and necrosis. The sensitivity, specificity, PPV, NPV, and overall accuracy of all biopsies were 89.1%, 100%,100%, 70.4%, and 91.3% respectively; The sensitivity, specificity, PPV, NPV, and accuracy for TRUS-guided biopsy and CE-TRUS guided biopsy were 73.7%, 100%,100%, 50%,79.2% and 92.1%,100%,100%,75%,93.6% separately; The increase in correct diagnoses was significant (p < 0.05) between TRUS guided biopsy and CE-TRUS guided biopsy.TRUS-guided biopsy is a reliable procedure that can be augmented by endoscopic biopsy techniques if the biopsy yields negative results. CE-TRUS might assist in the location of the biopsy and decrease sampling errors.© 2023. The Author(s).