Flower color is an important ornamental feature, often modulated by the contents of flavonoid. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoid, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, and classified into the cluster of bona fide CHSs. Thus RdCHS1 was then subjected for further functional analysis. Real-Time PCR analysis revealed that transcripts of RdCHS1 was the highest in leaves and lowest in root, did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. Physiological function of RdCHS1 was checked in Arabidopsis mutant and tobacco, the results showed RdCHS1 transgenes could recover the color phenotypes of tt4 mutant and made tobacco flower color from pink to dark pink through modulating the expression of endogenous structural and regulatory genes in tobacco. All these results demonstrated that RdCHS1 is a bona fide CHS and contribute to flavonoid biosynthesis in R. delavayi.