This article combines bioinformatics， quantitative real-time PCR， in situ hybridization， and HPLC techniques to investigate the sequence characteristics， spatiotemporal expression patterns， and subcellular localization of the Bco1 gene in the Chinese mitten crab （Eriocheir sinensis）. It also explores the variations in Es-Bco1 expression， color， and carotenoid content in the hepatopancreas under chronic stress caused by high pH levels. The findings reveal that the full-length of Es-Bco1 open reading frame （ORF） spans 1 614 bp and encodes 537 amino acids. It belongs to the RPE65 superfamily and exhibits a high similarity to BCO1 in the Portunus trituberculatus.（2） Es-Bco1 was expressed in multiple tissues， with high expression in the endoderm， eyestalks， and hepatopancreas； the expression of Es-Bco1 in the hepatopancreas was highest during the post-molt stage （stage AB）； the expression of Es-Bco1 in the hepatopancreas significantly increased during ovarian stages Ⅰ-Ⅲ and then significantly decreased. （3） In situ hybridization results showed that Es-Bco1 mRNA was mainly localized in the F cells （Fibrillar cell） and R cells （Resorptive cell） of the hepatopancreas during the AB stage and ovarian development stage Ⅲ. （4） After 30 days of exposure to pH 8.0 （control）， 9.0， and 10.0， the expression level of Es-Bco1 in the hepatopancreas of juvenile crabs significantly increased， while the lightness value （L^*）， yellowness value （b^*）， total carotenoid content， and β-carotene content in the hepatopancreas significantly decreased. In conclusion， Bco1 in E. sinensis is mainly expressed in the epidermal layer， eyestalks， and hepatopancreas， with high expression during the post-molt stage and ovarian stage Ⅲ. Furthermore， high pH stress can upregulate Es-Bco1 expression in the hepatopancreas and reduce carotenoid content， indicating that Bco1 plays an important role in carotenoid cleavage of E. sinensis in response to high pH stress.