<p>Quenchbodies (Q-bodies) are fluorescent antibodies that respond to antigen binding via a fluorescence quenching and de-quenching mechanism. To enhance the versatility of the generation method and expand the color range, we developed a novel Q-body generation approach using tyrosinase-mediated site-specific conjugation to a tyrosine-rich hemagglutinin (HA)-tag. A single-chain variable fragment (scFv) against programmed cell death-ligand 1 (PDL1) was engineered with an N-terminal HA-tag and expressed in <i>Escherichia coli</i> with high yield and purity. Site-specific conjugation of four hydrazide-functionalized dyes with different emission wavelengths was achieved using recombinant tyrosinase. The resulting Q-bodies were evaluated via fluorescence-linked immunosorbent assay, all of which demonstrated antigen concentration-dependent fluorescence enhancement. Notably, the tetramethylrhodamine&#xa0;(TAMRA)-labeled Q-body showed the highest signal-to-background ratio, limit of detection of 0.51 ± 0.01&#xa0;μg/mL. This HA-tag-mediated Q-body generation strategy offers a robust and versatile tool for the production of Q-bodies with broad dye compatibility, enabling sensitive and multiplexed fluorescent immunoassays.</p> Graphical Abstract <p></p>