The invention belongs to the technical fields of stem cell biology and cell culturing, and provides a serum-free culturing system inducing differentiation of pluripotent stem cells into motor neuron cells. The serum-free culturing system specially comprises a serum-free differentiation basic culture medium and an additive for promoting the differentiation of the motor neuron cells, wherein necessary components of the serum-free differentiation basic culture medium comprise a DMEM/F12 culture medium, a human serum albumin, L-glutamine, D-glucose, vitamin C, N2/B27 cell culture additive and unnecessary amino acid. The additive for promoting the differentiation of the motor neuron cells specially comprises SB431542, dermomorphin, fibroblast growth factors-2, retinoic acid, BDNF, GDNF, CTNF, DAPT, Smoothened agonist, and cytarabine. The culture medium is adopted to help improve the differentiation of the motor neuron cells in a cell mixing system, the differentiation of other types of cells is reduced, and finally, the motor neuron cells with high purity can be obtained.