The soybean isoflavone is a kind of widely used active materials which has medical and healthcare functions. To analyze the differential expression patterns of genes associated with isoflavone synthesis in soybean, seven related genes were studied at different developmental stages and organs in different varieties by real-time quantitative PCR method. The results showed that the expression levels of phenylalanine ammonialyase PAL, cinnamate-4-hydroxylase C4H and 4-coumarate: coenzyme A ligase 4CL in Zhongdou 27 (high isoflavone content variety) in the leaves were significantly higher than that in Chuxiu (low isoflavone content variety) during the R2 growing period. Meanwhile, the expression levels of chalcone synthase CHS and isoflavone synthase IFS in Zhongdou 27 were significantly higher than that in Chuxiu during the R8 growing period in the seeds. While the expression level of cytochrome P450 reductase CPR in Chuxiu was significantly higher than that in Zhongdou 27 during the R7 period in both of the leaves and seeds. Thus, these differential expression levels of related genes in different developmental stages and organs may be the main reasons to explain different seed isoflavone contents in soybean.