This invention provides method and test kit for synchronously detecting sub-genotypes and drug-resistant mutation of hepatitis B virus (HBV). The test kit comprises: two specific primers for amplifying gene or transcript of HBV, three specific oligonucleotide probes for detecting sub-genotypes of HBV, and thirteen specific oligonucleotide probes. The primers and the probes can also be replaced with the complementary sequences. The test kit can synchronously differentiate three sub-genotypes (B, C and D) of HBV, and detect lamivudine- and adefovir dipivoxil-resistant mutations. The test kit can reduce the detection cost.