Gene expression valuated by reverse transcription-quantitative PCR (RT-qPCR) are often applied to study the gene function. To obtain accurate and reliable results, the usage of stable reference genes is essential for RT-qPCR analysis. The traditional southern Chinese medicinal herb, Desmodium styracifolium Merr is well known for its remarkable effect on the treatment of urination disturbance, urolithiasis, edema and jaundice. However, there are no ready-made reference genes identified for D. styracifolium. In this study, 13 novel genes retrieved from transcriptome datasets of four different tissues were reported according to the coefficient of variation (CV) and maximum fold change (MFC) of gene expression. The expression stability of currently used Leguminosae ACT6 was compared to the 13 candidate reference genes in different tissues and 7-day-old seedlings under different experimental conditions, which was evaluated by five statistical algorithms (geNorm/NormFinder/BestKeeper/ΔCT/RefFinder). Our results indicated that the reference gene combinations of PP  +  UFM1, CCRP4  +  BRM and NFD6  +  NCLN1 were the most stable reference genes in leaf, stem and root tissues, respectively. The most stable reference gene combination for all tissues was CCRP4  +  CUL1. In addition, the most stable reference genes for different experimental conditions were distinct, for instance SMUP1 for MeJA treatment, ERDJ2A  +  SMUP1 for SA treatment, NCLN1  +  ERDJ2A for ABA treatment and SF3B  +  VAMP721d for salt stress, respectively. Our results lay a foundation for achieving accurate and reliable RT-qPCR results so as to correctly understand the function of genes in D. styracifolium. BACKGROUND The online version contains supplementary material available at 10.1007/s13205-021-02954-x.