We demonstrate the use of functional surface groups inherently present on off-stoichiometric thiol-ene polymers, for site-specific immobilization of biomolecules and detection by evanescent wave-induced fluorescence. An optofluidic chip featuring an embedded thiol-ene waveguide was selectively functionalized with biotin using photografting. The biotin was used for immobilization of fluorescently labelled streptavidin, and experiments revealed a linear correlation between streptavidin concentration and fluorescent intensity. To further demonstrate the attractiveness of using thiol-ene for optofluidic devices, the optical properties of thiol-ene was evaluated by determining the transparency and refractive index of the cured polymer.