Microalgae have been considered as a potential candidate for biodiesel feedstock. Single-stage simultaneous extraction–transesterification process is proposed for simpler and more effective biodiesel conversion. In this study, the experiment of biodiesel production from microalgae Spirulina sp. was performed in a batch-stirred reactor using palm oil as a co-solvent of methanol and catalyzed by potassium hydroxide at a percentage of 1 wt% (w/w of palm oil). The effects of methanol–palm oil molar ratio, palm oil–microalgae weight ratio, and temperature on biodiesel yield were investigated. The results showed that the best biodiesel yield was 85.28% (99.01% of partial biodiesel yield from palm oil and 16.69% of partial biodiesel yield from dry microalgae), obtained at a methanol–palm oil molar ratio of 10:1, a palm oil–microalgae weight ratio of 5:1, and at a temperature of 60°C. Upon comparison, the overall yield increased by 34.59% (37.73% of partial biodiesel yield from palm oil and 13.00% of partial biodiesel yield from dry microalgae) than that of the two-stage (conventional) method. Single-stage simultaneous extraction–transesterification process also reduced the number of unsaturated fatty acid components in biodiesel that will lower the biodiesel quality.