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This paper reports on our study using several optical filters known to be efficient in separating compounds having various levels of maximum luminescence, to separate information from three kinds of Luciola lateralis luciferase with a maximum luminescence of 559 nm, 604 nm and 607 nm. Simultaneous luminescence of Luciola lateralis luciferase was determined by measuring the luminescence through a band pass filter or sharp cut filter (BPB50, 53, 58, No.58, SC58, 60, 62, 64). It was possible to determine luciferase with a maximum luminescence lambda(max) of 559 nm (yellow-green) utilizing the band pass filter (BPB 50), described here. Meanwhile, luciferase with a lambda(max) of 607 nm (red) could be determined by calculations based on the bioluminescent intensity through the band pass filter and sharp cut filter (SC58). In addition, we also applied a simultaneous bioluminescent enzyme immunoassay of pepsinogen I (PGI) and pepsinogen II (PGII) in which two kinds of biotinylated luciferase (Luciola lateralis) labelled as an enzyme producing yellow-green light (lambda(max) = 559 nm) and red light (lambda(max) = 607 nm) were used. In the proposed method, PGI and PGII in serum were simultaneously captured in a sandwich-type immune reaction between anti-PGI and anti-PGII monoclonal antibody-coated magnetic particles, and streptavidin-biotinylated luciferase biotinylated anti-PGI and anti-PGII monoclonal antibodies triplexes, respectively. The result was a calibration range for PGI of 2-200 ng/mL, and for PGII of 1-100 ng/mL. In conclusion, the correlation of PG values in serum between the proposed method (simultaneous assay) and an individual specific bioluminescent immunoassay (specific assay) were satisfactory.