On the effect of substrate bias at first stage of diamond synthesis at lower substrate temperature(approximately 673K) using microwave plasma CVD and effect of reaction gas system for the bias enhanced nucleation were studied. The reaction gas was mixture of methane and hydrogen or carbon monoxide and hydrogen. The nucleation density of applied bias -150V using $CH_4-H_2$ reaction gas system, significantly higher than that of $C-H_2$ reaction gas system. When the $CH_4-H_2$ reaction was used, nucleation density was increased because of existence of SiC as a interface for diamond nucleation. By use of this negative bias effect for fabrication of CVD diamond film using two-step diamond growth without pre-treatment, fabrication of the diamond film consist of diamond grains $0.2mu extrm{m}$ in diameter was demonstrated