SU-8 is an attractive platform for the development of smart biochips owing to its high aspect ratio of micro/nanostructures fabrication and remarkable optical and biocompatible properties. However, few works have explored sub-micron SU-8 thin films for applications in new generations of portable bioanalytical devices. In this work we discuss surface properties for the efficient immobilization of different bioanalytical components on SU-8 thin film surfaces. Short exposure time oxygen plasma treatment improved hydrophilicity and activation of surface carboxyl groups of 300 nm-thick SU-8 films, while maintaining surface roughness below 2 nm. Under these optimized surfaces conditions, covalent immobilization of Interleukin-6 and Prostate Specific Antigen antibodies on SU-8 surfaces was evaluated using quantitative fluorescence microscopy. The addition of standard crosslinker, 1-ethyl-3-(3-(dimethylamino)-propyl)-carbodiimide and N-hydroxysuccinimide mixture to our protocol yielded 15–20% higher antibody immobilization due to activation of surface carboxyl groups. The stability of the plasma treatment along time, and the ideal surface passivation of functionalized samples are demonstrated, along with the selective immobilization antibody to photolithographically-patterned SU-8 microstructures. Overall, our optimized protocol could find broad applications of functionalized SU-8 thin films in biomedical fields, particularly for the fabrication of SU-8 based miniaturized photonic and plasmonic biosensors.