BACKGROUND: The operation sequence of bidirectional endoscopy with topical anesthesia varies among endoscopists, which interferes with clinical procedure. The study aimed to investigate the influence of different operation sequences on the outcomes of quality indicators and improve bidirectional endoscopy performance. METHODS: A randomized trial was conducted at the affiliated hospital of a medical university in China. Outpatients who initially underwent bidirectional endoscopy with topical anesthesia were enrolled. Eligible patients were randomized to either the colonoscopy-first group or the esophagogastroduodenoscopy-first group. Dyclonine hydrochloride mucilage and oxybuprocaine hydrochloride gel were administered for topical anesthesia. After finishing the previous process, the subsequent one was performed immediately. Quality indicators of esophagogastroduodenoscopy and colonoscopy were compared between the groups. RESULTS: Analyzing 395 combined procedures, the cecal intubation rate, discomfort score during esophagogastroduodenoscopy, examination score of esophagogastroduodenoscopy, and colorectal polyp detection rate were similar between the two groups. The colonoscopy-first group had lower colonic spasm incidence (66.0% vs. 30.3%, p <
0.001), shorter median cecal intubation time (254 s vs. 211 s, p <
0.001), and higher colonoscopy comfort rate (72.5% vs. 85.6%, p <
0.001) compared with the esophagogastroduodenoscopy-first group. Operation sequence significantly affected the incidence of colonic spasm (OR 4.739, 95%CI 3.054-7.352, p = 0.000), which correlated with cecal intubation time (r = 0.196, p <
0.001) and patient discomfort score (r = 0.136, p = 0.007). CONCLUSION: In bidirectional endoscopy with topical anesthesia, performing colonoscopy first may improve colonoscopy performance without affecting esophagogastroduodenoscopy examination. The study was registered prior to conducting the research in the Chinese Clinical Trial Registry ( https://www.chictr.org.cn ) on November 7, 2023 with the trial identification number ChiCTR2300077408.