The invention discloses a hepatitis B virus lamivudine resistant RNA quantitative detection kit, a detection method, primers and probes. The kit comprises fluorescent quantitative polymerase chain reaction (PCR) solution which contains diethyl pyrocarbonate (DEPC) treatment water, DNA polymerase with 5'-3' exonucleolytic activity, dNTPs, 10X fluorescent quantitative PCR Buffer, solution containing Mg<2+>, rt204 locus forward primer, a rt204 locus backward primer, a rt204M locus probe, a rt204I locus probe, a rt204V locus probe, a rt180 locus forward primer, a rt180 locus backward primer, a rt180L locus probe and a rt180M locus probe. The kit also comprises DNA extraction solution, a negative control, a working standard, a positive control and a critical positive control. In the invention, a real-time fluorescent quantitative PCR technique is adopted to quantitatively detect a lamivudine resistant hepatitis B virus strain, the fluorescent quantitative PCR method is changed into a '2-step method', and the step of PCR extension is avoided; and the probes can mark various fluorescent marks and have the advantages of specificity, sensitiveness, quickness and simple and convenient operation.