While studying the mechanism of ginsenoside Rg3 (G-Rg3) on tumor inhibition, we produced monoclonal antibody to G-Rg3 for more specific investigation. We immunized Balb/c mice to G-Rg3 conjugated bovine serum albumin (BSA) by intraperitoneal injection and hybridized splenocytes from those immunized mice and myeloma cells. From those fusion cell lines, we selected productive monoclonal clones and obtained culture media containing monoclonal antibody to G-Rg3. After purification, we performed enzyme-linked immunosorbent assay (ELISA) to verify the sensitivity and specificity of the antibody. When compared with G-Rh2 having a very similar structure as a metabolite of G-Rg3, the antibody worked only with G-Rg3 in a concentration-dependent manner. We confirmed that the monoclonal antibody to G-Rg3 can be applied to immunocytochemistry for detection of the treated G-Rg3 inside A549 human lung adenocarcinomas. Thus, the monoclonal antibody to G-Rg3 would be a useful tool for measuring the bioactivity of G-Rg3 in various fields.