Abstract: For further promoting the obtaining of NLP gene mutant soybean materials and studying the functions of GmNINs and GmNLPs, this study used bioinformatics methods to analyze GmNINs and GmNLPs, and constructed the knockout mutation vectors by CRISPR/Cas 9 technology, and then verified the effectiveness of the targets by soybean hair root transformation experiments. A total of 4 GmNINs and 10 GmNLPs were obtained by bioinformatics analysis, and each had two conserved domains, RWP-RK and PB1, and conserved gene structure. Subcellular localization prediction indicated all these NIN and NLP members were located in the nucleus, and GmNIN1 b and GmNIN2 a are also located in chloroplasts. In nodules, the expression of GmNIN1 a/b, GmNIN2 a/b, GmNLP2 a/b and GmNLP3 b was relatively high, predicting that these genes may have important functions in nodules. Two knockout vectors NLP4 and NLP5 were successfully constructed, and three effective targets for knocking out GmNLP4 a/b and two effective targets for knocking out GmNLP5 a/b were obtained. This study provides a biological information basis for the research of soybean NIN and NLP family gene, and also provides technical support for the mutants of soybean NLP4 a/b and NLP5 a/b genes.