An ordered adsorption orientation is significant for the catalytic activity of immobilized enzymes. In this study, the orientations and conformation of ribonuclease A (RNase A) adsorbed on oppositely charged self-assembled monolayers (SAM) have been studied by a multiscale approach, including parallel tempering Monte Carlo, all-atom molecular dynamics and coarse-grained molecular dynamics simulations. Simulation results show that RNase A adsorbed on oppositely charged surfaces with opposite orientations. The active site of RNase A is oriented toward the surface when it adsorbs on a negatively charged surface; while for RNase A adsorbed on a positively charged surface, the active site is oriented toward the solution. Negatively charged surfaces could be used for RNase A removal since the catalytic active site is blocked. To bring the enzymatic catalysis of RNase A into play, positively charged surfaces can be used to control the orientation of RNase A with the active site accessible. The dipole moment and side chains of RNase A on both surfaces are slightly changed, whereas the backbone structure of RNase A is well preserved. That is to say, RNase A preserves its native conformation during the adsorption process. The simulation results could be applied into the design and development of substrates for the immobilization of ribonuclease (C) 2014 Elsevier Ltd. All rights reserved.