A gene sequencing substrate and a method for manufacturing the same, and a gene sequencing device are provided. It belongs to the technical field of gene sequencing, and can solve the problem of high cost of the high-throughput sequencing chip in the prior art. The gene sequencing substrate of the present disclosure comprises a plastic material with concave structures as base substrate, and the concave structures serve as reaction cells. Since the base substrate has plasticity, the concave structures can be formed by a simple process to reduce the cost of the gene sequencing substrate. Meanwhile, a first protective layer may be provided on the inner wall of the concave structures for preventing the inner wall of the concave structures from being corroded by the reaction liquid.