New fusion proteins for immunoassay were developed as an alternative to the conventional chemical linked enzyme-antibody complex. Human chimeric alkaline phosphatase (IPP) was used as the labeling enzyme, and partial domains of Staphylococcus aureus Protein A and Streptococcus Protein G were used as antibody binding protein (PG). The fusion protein composed of IPP and PG was produced using human cell lines because IPP is derived from human enzymes. The expression system was optimized by changing the plasmid vector, reducing the GC content of the DNA sequence, and employing different cell lines including adherent and suspension cells. As result, a 2,600-fold increase in the protein yield per unit of growth medium was achieved. The resultant IPP-PG fusion protein was successfully utilized for immunoassay applications such as western blotting and immunohistochemistry.