Product defects in an assembly line happen due to various reasons, and one of them is human error. The occurrence of human errors in manual assembly line can be affected by workplace condition and environment, equipment as well as demographics factors. The aim of this research was to investigate the effect of assembly line parameters such as time pressure, working position, jig design, and component bin position; and gender on human errors during the manual assembly. An experiment based on full factorial design was conducted in the lab, where three-pin plug assembly was selected to simulate the assembly task. A total of ten participants were involved. The results showed that among all the parameters studied, time pressure had the most significant effect on human error, followed by working position and gender difference. Meanwhile, the jig design had no effect on human error but gender difference had contributed towards the differences on human error, where females made fewer errors than males.