In this work, surface-enhanced Raman scattering (SERS) was applied to probe the orientation of cytochrome c (Cyt-c) on gold nanohole arrays functionalized with self-assembled monolayers (SAMs) of alkane thiols with positively (-NH2) and negatively (-COOH) charged terminal groups. Square grid gold nanohole arrays with a nanohole diameter of 270 nm and a grating of 350 nm were fabricated by electron beam lithography (EBL) and were used as the SERS substrates. The SERS intensities of the nontotally symmetric mode (B(1g) mode nu(11)) and the totally symmetric mode (A(1g) mode nu(4)) and their ratios were used to determine the orientation of Cyt-c on surfaces. The results indicate that the heme group is close and perpendicular to the negatively charged surface but is far from and oriented at an angle to the positively charged surface. Cyt-c has a random or more flat orientation on the bare Au nanoholes surface.