In this paper, we further investigate the properties of off-stoichiometry thiol-ene polymers (OSTE) aiming its application in microchip electrophoresis for bioanalytical applications. The proportion of 1.3:1 (allyl:thiol) and 1:2.5 (allyl:thiol) presented the best results in terms of sealing. Raman imaging mapping of the polymers surfaces revealed an outstanding homogeneity. Water contact angle were measured as 68° ± 6° and 71° ± 5° for 1.3:1 allyl and 1:2.5 thiol, respectively. Substrates prepared with OSTE demonstrated to be less prone to sorption of nonpolar compounds. The electroosmotic flow measured for this OSTE composition was 3.8 ± 0.3·10-4 cm2 s-1 V-1, 1.5 times higher than the one found for polydimethylsiloxane microchips under the same conditions. As a proof-of-concept for the applicability of OSTE microchips in bioanalysis the immobilization of α-amylase on the polymer surface and the implementation of a Saccharomyces cerevisiae cell counter using contactless conductivity detection are demonstrated.