The invention relates to the technical field of molecular biology, in particular to Indexes for multiplexed nucleic acid sequencing and PCR primers for constructing 16SrRNA multiplex nucleic acid sequencing libraries. According to the Indexes provided by the invention, a sequence difference degree between the indexes, a base identification rate and preference of a sequencing reaction on data of various Index sequences are taken into full consideration, and when the indexes are included in the sequencing or library-building primers, sequencing data obtained from a hybrid sequencing reaction is relatively good in balancing performance. The PCR primers provided by the invention are quite suitable for a soil sample, and the PCR primers are strong in specificity, clear in target amplification bands and free from a dispersion phenomenon, so that a research on bacterial diversity in the soil sample is more efficient, convenient and accurate. The PCR primers provided by the invention contain the Indexes provided by the invention and an obtained amplification product has two Indexes; and when the Indexes are applied to hybrid sample sequencing, different sequencing libraries can be distinguished.