The orientation of antibodies on surfaces is critical to the performance of biosensors. Due to the charge distribution within an antibody molecule, it is hoped that the orientation of adsorbed antibodies can be controlled by adjusting microenvironments (e.g., surface and solution properties). In this work, we investigated the orientations of two types of monoclonal anti-human chorionic gonadotropin (anti-hCG) adsorbed on NH2 (positively charged) and COOH (negatively charged) terminated self-assembled monolayers (SAMs) by surface plasmon resonance (SPR) biosensors. Adsorbed antibody molecules on surfaces were also characterized by atomic force microscopy. Results show that the orientation of the IgG1 type antibody is better on the NH2 surface than on the COOH surface, as indicated by a higher hCG to adsorbed anti-hCG ratio on the NH2 surface from SPR experiments. However, for the IgG2a type antibody, the antigen-to-antibody ratio, and thus the orientation of the adsorbed IgG2a, is similar on the NH2- and COOH-terminated SAM surfaces. Surface charge affects the orientation of IgG2a less than IgG1 due to the smaller dipole moment of IgG2a compared to IgG1. This work provides not only a fundamental understanding of how microenvironments affect protein behavior on surfaces but also a useful guide to designing surfaces for applications, such as biosensors and biomaterials.