The invention discloses a single-molecule gene sequencer, and belongs to technical schemes of the field of gene sequencing. The single-molecule gene sequencer comprises a base, wherein a clamping platform, a reagent storing device, a fluid control device, a moving platform and a total-reflection microscope are arranged on the base; a gene sequencing chip is arranged on the clamping platform; the reagent storing device is used for storing a gene sequencing reagent; the fluid control device is used for pumping the gene sequencing reagent into the gene sequencing chip from the reagent storing device; the moving platform is used for driving the clamping platform to move to or away from the part below the a total-reflection microscope; the total-reflection microscope is used for detecting gene sequences of samples in the gene sequencing chip. The scheme has no library development flow or PCR, is simple and convenient to operate and low in sequencing cost, and is a diagnosis and treatment method very applicable to clinical application.