Intelectin is a recently characterized soluble galactofuranose-binding lectin that exists in species ranging from amphioxus to human. Interestingly, intelectin does not contain a canonical carbohydrate-recognition domain (CRD). Therefore, we designed serial deletions of intelectin in the Chinese amphioxus (Branchiostoma belcheri tsingtauense, AmphiITLN71469) in order to identify functional regions required for carbohydrate binding. Our results revealed that Domain 5 (aa 203–302) was able to bind lipopolysaccarides (LPS) or peptidoglycan (PGN) and agglutinate bacteria as efficiently as the full-length protein. Three dimensional (3D) atomic models of Domain 5 were generated by ab initio based program QUARK and by Iterative Threading Assembly Refinement (I-TASSER) programs, in which four amino acids mediating calcium-binding (G54-G55-G56-E91) were identified by hemagglutination assay. Furthermore, a striking functional conservation of Domain 5 was detected in zebrafish intelectin 1. Taken together, our findings identified for the first time a new CRD domain in intelectin, thereby providing new knowledge leading to a better understanding of pathogen-host interactions.