Nanomaterials without chemical linking or physical interaction resided on two-dimensional surface have been attractive due to their unique quantum confinement properties and further electronics and biomedical applicability. An in situ method has been developed to fabricate gold nanoparticle (NP) films on different substrates regardless of hydrophilic or hydrophobic surfaces, including glass, 96 polystyrene well plates, and polydimethylsiloxane (PDMS). A mixture of HCOONa and HAuCl4 solution was used to obtain Au NP films at room temperature on the hydrophilic and hydrophobic surfaces. Experimental study of the mechanism revealed that film formation is dependent on surface wettability. The surface-enhanced Raman scattering enhancement grew stronger on films obtained with solutions containing increasing sodium formate (HCOONa) concentrations. The as-fabricated Au NP films were also used to successfully bind antibodies to check their applicability in bioassays.