Valerate-hydrolysate enriched culture showed great ability to produce 3HV and 3H2MV. Valerate-hydrolysate enriched culture had more in the community. Mixed iso-/n-valerate was effective at balancing microbial growth and PHAs synthesis. Co-substrates of valerate and propionate raised the fraction of 3HV and 3H2MV. The production of polyhydroxyalkanoates (PHAs) with a high fraction of 3-hydroxyvalerate (3HV) and 3-hydroxy-2-methylvalerate (3H2MV) from mixed culture enriched by valerate-dominant hydrolysate was evaluated in this study. After long-term enrichment, the culture showed strong ability to synthesize 3HV and 3H2MV, even with acetate-dominant substrate. The ultilization of single or mixed iso-/n-valerate by the enriched culture showed that the mixture of iso-valerate and n-valerate was more efficient substrate than any single in terms of balancing microbial growth and PHAs synthesis. Besides, through comparing the kinetics and stoichiometry of the tests supplying valerate and propionate, the enriched culture with equivalent valerate and propionate (1:1 molar ratio) exhibited superior PHAs production performances to pure valerate or propionate, attaining more than 70 mol% of 3HV and 3H2MV. The above findings reveal that valerate-dominant hydrolysate is a kind of suitable substrate to enrich PHAs producing culture with great capability to synthesize 3HV and 3H2MV monomers, thus improving product properties than pure poly(3-hydroxybutyrate) (P3HB); also 3HV and 3H2MV production behaviors can be regulated by the type of odd-carbon VFAs in the substrate.