Preparation of mouse embryonic fibroblast (MEF) feeder cells to maintain pluripotent stem cells (PSCs) is time consuming and involved in animal issues. Here, we demonstrated a novel method to prepare feeder cells with high efficiency, timesaving, and low costs. MEFs in 3 × 104 cell/cm2 were fixed by methanol for 5 min and air drying for 5 min. Thereafter, the methanol fixed MEF cells (MT-MEF) were able to be used directly to culture PSCs or stored at room temperature for the future usage. PSCs cultured on MT-MEF could be continuously expanded for over 40 passages with the naïve pluripotency. MT-MEFs could also be used to maintain human and pig iPSCs. Moreover, methanol fixed MEFs’ culture dish was able to be reused for at least 4 times, and to be applied for antibiotic resistant screening assay to establishing stable transfected PSC lines. Alternatively, the immortalized cell lines, for instance NIH3T3 cells, could also be fixed by methanol and used as feeder cells to maintain PSCs. Thus, this novel means of methanol fixed feeder cells can completely replace the mitomycin C and gamma radiation treated MEF feeder cells, and be used to maintain PSCs derived from mouse as well as other animal species.