Pemphigus vulgaris is a severe autoimmune disorder resulting from production of anti-desmoglein 3 (Dsg3) antibodies. An accessible standardized antigenic component, the recombinant Dsg3 extracellular component, is necessary to develop domestic diagnostic ELISA kits. The retrospective experimental study aimed to produce a recombinant Dsg3 ectodomain (ЕС1-ЕС5 domain) in the heterologous expression system and assess its immunoreactivity with autoantibodies of patients having pemphigus vulgaris when used as a target antigen in ELISA. The synthetic gene encoding the Dsg3 ЕС1-ЕС5 domains was cloned into eukaryotic expression vectors p1.1-Tr2 and p1.2-GS. The resulting constructs were used for transfection of CHO-4BGD cells. The target protein was purified by affinity chromatography on the Ni-Sepharose FF sorbent. Immunoreactivity of the recombinant Dsg3 as an antigenic substrate was assessed by ELISA using sera from patients with pemphigus vulgaris (n = 12) and healthy donors (n = 5) under two buffer conditions: with EDTA (Dsg3-EDTA) and with added Ca²⁺ ions (Dsg3-Ca²⁺). The Euroimmun commercially available ELISA kit (target antigen: Dsg3 ectodomain) was used for reference. As a result, transient cultures of the cells producing the Dsg3 ЕС1-ЕС5 domains were obtained. The protein with the purity > 90% was isolated by affinity chromatography. When performing ELISA in the Dsg3-EDTA buffer,  33.3% of samples were classified as positive, while 50.0% were considered positive when using the Dsg3-Ca²⁺ buffer; the effect of adding Ca²⁺ was heterogeneous: 58.3% of samples showed the signal increase and 41.7% showed the decrease. The findings suggest the need to switch to the Dsg3 expression in human cells (HEK293).