Objective: To verify the feasibility and accuracy of the transanal multipoint full-layer puncture biopsy (TMFP) technique in determining the residual status of cancer foci after neoadjuvant therapy (nCRT) in rectal cancer. Methods: Between April 2020 and November 2022, a total of 78 patients from the Beijing Chaoyang Hospital of Capital Medical University, the Beijing Friendship Hospital of Capital Medical University, the Qilu Hospital of Shandong University, the Zhongnan Hospital of Wuhan University with advanced rectal cancer received TMFP after nCRT participated in this prospective multicenter trial. There were 53 males and 25 females, aged (M(IQR)) 61 (13) years (range: 35 to 77 years). The tumor distance from the anal verge was 5 (3) cm (range: 2 to 10 cm). The waiting time between nCRT and TMFP was 73 (26) days (range: 33 to 330 days). 13-point transanal puncture was performed with a 16 G tissue biopsy needle with the residual lesion as the center. The specimens were submitted for independent examination and the complications of the puncture were recorded. The consistency of TMFP and radical operation specimen was compared. The consistency of TMPF with clinical remission rates for the diagnosis of complete pathological remission was compared by sensitivity, specificity, negative predictive value, positive predictive value and accuracy. Statistical analysis between groups was performed using the χ2 analysis, and a paired χ2 test was used to compare diagnostic validity. Results: Before TMFP, clinical complete response (cCR) was evaluated in 27 cases. Thirty-six cases received in vivo puncture, the number of punctures in each patient was 13 (8) (range: 4 to 20), 24 cases of tumor residue were found in the puncture specimens. The sensitivity to judgment (100% vs. 60%, χ2=17.500, P<0.01) and accuracy (88.5% vs. 74.4%, χ2=5.125, P=0.024) of TMFP for the pathologic complete response (pCR) were significantly higher than those of cCR. Implement TMFP based on cCR judgment, the accuracy increased from 74.4% to 92.6% (χ2=4.026, P=0.045). The accuracy of the in vivo puncture was 94.4%, which was 83.3% of the in vitro puncture (χ2=1.382, P=0.240). Overall, the accuracy of TMFP improved gradually with an increasing number of cases (χ2=7.112, P=0.029). Conclusion: TMFP is safe and feasible, which improves the sensitivity and accuracy of rectal cancer pCR determination after nCRT, provides a pathological basis for cCR determination, and contributes to the safe development of the watch and wait policy.