Chitinase plays a role in mammalian immune responses, particularly in the degradation of fungal cell walls. The aim of the present study was to express and characterize recombinant mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) without the ZZ domain, a domain that may interfere with immunological analyses. We successfully expressed recombinant chitinases without the ZZ domain (Chit1-V5-His and AMCase-V5-His) as a soluble protein from an expression vector pET21a in the Escherichia coli Rosetta-gami B (DE3) strain. Chit1-V5-His exhibited chitinolytic activity similar to that of ProteinA-Chit1-V5-His (a recombinant Chit1 with the ZZ domain) and natural Chit1, both with synthetic and natural substrates. Differential scanning fluorimetry and thermal stability assays revealed that Chit1-V5-His retained functional stability comparable to that of ProteinA-Chit1-V5-His, although ProteinA-Chit1-V5-His was more thermally stable. AMCase-V5-His demonstrated prominent chitinolytic activity at pH 2.0, aligning with the properties of natural AMCase. Owing to the lack of the ZZ domain that potentially binds to immunoglobulin G Fc region, Chit1-V5-His and AMCase-V5-His are advantageous tools for immunological analyses, as they do not block the Fc receptor-mediated phagocytosis of fungi by polymorphonuclear neutrophils and macrophages. Thus, this expression system effectively produces functional chitinases, facilitating further studies on their roles in mammalian immunity.