Bias voltage applied to the substrate holder during thin film deposition is frequently used for modifying the coating properties in magnetron sputtering process. In this work, we have studied the indentation properties of sputter deposited Cu thin films on single crystal Si substrates under different bias in an effort to understand the effect of bias on the indentation properties and microstructure of soft films on hard substrates. The bias voltage was varied in the range 40 V to 100 V. The deposited thin films were characterized by transmission electron microscope (TEM), X-ray diffraction (XRD) and nanoindentation technique. The results show that with the bias voltage increasing, the grain size and residual stress decrease, which induce the hardness of Cu thin film increasing and it reaches the maximum at 80 V and then it decreases. At the same time, the indentation size effect has great influence on the hardness with depth increases.