A novel route was introduced to synthesize dense polyacrylamide (PAM) onto the glass slide surface. To investigate the surface chemistry of the PAM on the glass slides, X-ray photoelectron spectroscopy (XPS) was utilized to obtain detailed chemical state information on the PAM layer constituents. The XPS peak data were consistent with the presented model of the PAM on the glass slide surface. Scanning electron microscopy and atomic force microscope data indicated the presence of PAM on the glass slides, which consist of nodules. The results showed that PAM was successfully immobilized onto glass slides with a two-tier structure under aqueous condition and a monolayer structure under anhydrous condition. Compared with those under aqueous condition, the controllability of the molecular layer on glass slides and the reproducibility under anhydrous condition were much better, which makes anhydrous condition an advisable condition for the study of the reaction mechanisms of glass slides modified by PAM.