Significance The ability to simultaneously detect and identify multiple biological particles or biomarkers is one of the key requirements for molecular diagnostic tests that are becoming even more important as personalized and precision medicine place increased emphasis on such capabilities. Integrated optofluidic platforms can help create such highly sensitive, multiplexed assays on a small, dedicated chip. We introduce a method for multiplex fluorescence detection of single bioparticles by creating color-dependent excitation spot patterns from a single integrated waveguide structure. Detection and identification of individual virus particles from three different influenza subtypes are demonstrated. The principle can be readily applied to amplification-free detection of nucleic acid biomarkers as well as larger target numbers using combinatorial color coding.