High-precision register control is essential for manufacturing reliable micro-electronic devices using roll-to-roll (R2R) printing press. Thus far, many researches have reported on the structure of register controller and various control techniques, but printing defects in register marks have not been seriously considered even though it degrades the control performance by causing register measurement errors. This paper presents the statistical analysis on smearing defects according to printing conditions including ink viscosity, web speed, and doctor pressure. It analyzes the effects of the printing conditions on the mean and standard deviation of the smearing length using analysis of variance (ANOVA) and derives optimal printing conditions to minimize the smearing length using a Box-Behnken design. The effect of the optimization on the register measurement error was verified experimentally. The experimental results showed that the measurement and repeatability errors in the register measurement were improved by 73.3 and 42.3%, respectively, under the optimal conditions. This study suggests that the smearing defects could cause the register error and degrade the quality of printed layers, and the register error owing to the printing defects could be improved by changing parameters of printing press (e.g., web velocity and doctor pressure) as well as ink characteristics.