Objective: To select suitable references genes of Bupleurum scorzonerifolium for tissue expression analyses, and study the tissue expression characteristics of the key enzyme genes of saikosaponins biosynthesis. Methods: Five candidate reference genes including Actin, α-tubulin, β-tubulin, Cyclophilin, and EF-1α were chosen. The stability of these candidate reference genes was investigated by using four softwares (Delta CT, BestKeeper, NormFinder, and GeNorm). The stability of these candidate reference genes was tested and verified by real-time quantitative PCR. Used the stable reference gene, the tissue expression characteristics of the saikosaponins biosynthesis key enzyme genes (HMGR, IPPI, FPS, SS, and β-AS) was analyzed by qRT-PCR. Results: The average expression stability of the five candidate reference genes from high to low was β-tubulin > Cyclophilin > Actin > EF-1α > α-tubulin. Β-tubulin was the most suitable reference gene for tissue expression analysis in B. scorzonerifolium. HMGR expression level was roots > stems and fruits > leaves, IPPI expression level was roots > stems > fruits and leaves, FPS expression level was leaves > roots > stems and fruits, SS expression level was leaves > fruits > roots > stems, β-AS expression level was leaves > roots > fruits > stems. HMGR was significant positive correlated with IPPI, and FPS was significant positive correlated with β-AS (P < 0.05). Conclusion: β-tubulin gene was confirmed as the most suitable reference gene in different tissues of B. scorzonerifolium. It provided a methodological basis for the tissue expression analysis on the functional genes of B. scorzonerifolium. The expression pattern of five key enzyme genes of saikosaponins biosynthesis in different tissues had obvious differentiation, which might be involved in regulating the flow of saikosaponins synthesis and accumulation in various tissues of B. scorzonerifolium.