The invention relates to a fluorescent quantitative PCR detection kit and a detection method of HBV and TP of donor corneas. The kit comprises a DNA (deoxyribonucleic acid) extraction reagent, a DNA amplification reagent and positive working standards, wherein the DNA amplification reagent comprises a premix reagent, a 20X fluorescent probe reinforcing agent, forward and reverse HBV primers, an HBV fluorescent probe, forward and reverse TP primers, a TP fluorescent probe, distilled water of ribose-free nuclease and HBV and TP negative control and positive control; the positive working standards are pUC57-HBV plasmids containing destination sequences amplified by the forward and reverse HBV primers and a pUC57-TP plasmids containing destination sequences amplified by the forward and reverse TP primers. The invention realizes the complete closed-tube detection on donor conjunctiva tissue specimens and does not need PCR aftertreatment, and thereby, cross contamination is avoided; meanwhile, real-time detection is adopted, and an obtained Ct value and the original template number are completely in linear correlation; the quantitative accuracy rate is extremely high, and the relative error is about 50 percent, and thereby, the invention is enough to adapt to the demands of clinical nucleic acid quantification.