Objective To explore the value of endoscopic ultrasonography-guided carbon nanoparticles tattooing for preoperative localization of laparoscopic surgery for rectal cancer. Methods Sixty patients diagnosed as having rectal cancer who underwent laparoscopic radical resection in the 900th Hospital of Joint Logistics Support Force from April 2018 to April 2019 were randomly divided into the endoscopic ultrasonography-guided tattoo group (group A), the colonoscopy-guided tattoo group (group B) and the control group (group C) by random number table. The leakage rate of dye in intestinal tract, complications, and the tattooed serosa stained with carbon nanoparticles during laparoscopic surgery were recorded. Identification time of lesions, the total operation time, the length of resected rectal segment, tumor distance to distal resection margin, and the rate of anal preservation were analyzed and the postoperative pathology were compared. Results In group A, endoscopic ultrasonography revealed 2 cases of suspected tumor extension toward the anal side within distal intestinal wall. The extension length was 0.42 cm and 0.71 cm respectively, and the extension length was 0.36 cm and 0.64 cm under microscope respectively. In group B, the pathology result showed that the extension length was 0.53 cm under microscope. In group C, the pathology result showed that the extension length were 0.43 cm, 0.36 cm and 0.28 cm under microscope respectively. Obvious black staining in the rectal serosa was found in all tattooed patients during the surgery. There were scattered black staining spots at the surface of peritoneum and mesentery in 3 cases in group B. The identification time of lesions in group A and B were shorter than that in group C (1.29 ± 0.87 min, 1.31 ± 0.63 min VS 15.3 ± 10.50 min, P<0.05). The total operation time in group A and B were shorter than that in group C (176.12 ± 27.64 min, 175.67 ± 26.48 min VS 198.65 ± 38.67 min, P<0.05). The length of resected rectal segment in group A and B were shorter than that in group C (11.81 ± 5.76 cm, 12.31 ± 3.94 cm VS 15.24 ± 4.12 cm, P<0.05). The tumor distance to distal resection margin in group A and B were shorter than that in group C (3.61 ± 1.26 cm, 4.57 ± 1.58 cm VS 6.13 ± 2.47 cm, P<0.05). Anal preservation rates of three groups were 65.0% (13/20), 60.0% (12/20), 40.0% (8/20) respectively with no significant difference (P>0.05). No residual tumor cells were found in any specimens. Conclusion Carbon nanoparticles tattooing guided by endoscopic ultrasonography could reduce unnecessary intestinal segment incision and shorten the operation time. It can also reveal tumor extension toward the anal side within intestinal wall, which provides more accurate localization for the distal incision of the lower rectal cancer.