Conservation and sustainable management of biodiversity and ecosystem services are one of the major challenges faced by the world today. However, there is still limited research on how to identify and optimize the portfolio of priority conservation areas (PCAs) that integrate ecosystem services and biodiversity as conservation targets. Using the Three Parallel Rivers Region (TPRR) as a case, this study selected representative conservation features for both biodiversity and ecosystem services; biodiversity conservation features included the rare, endangered and endemic plant and animal species and natural vegetation types; ecosystem-service conservation features included regulation services (carbon storage, carbon sequestration and soil retention), cultural services (natural recreation) and supply services (water supply). We firstly identified the individual portfolio of PCAs for biodiversity and each type of ecosystem services by applying a systematic conservation planning approach, respectively. Then, we analyzed the relationship between these different portfolios of PCAs. We identified the PCAs by integrating biodiversity and ecosystem services that had a portfolio of PCAs being positively correlated with biodiversity PCAs. Finally, we assessed the conservation status of the integrated PCAs within six categories of the existing protected areas. The results showed that:(1) there was a positive relationship between different portfolios of the PCAs identified for biodiversity and each type of ecosystem service in TPRR; (2) In comparison with planning scenarios for ecosystem services and biodiversity separately, the PCAs integrating ecosystem services and biodiversity could simultaneously provide the highest coverage for all biodiversity features and ecosystem services; (3) The PCAs integrating ecosystem services and biodiversity covered 48.9% of the study area, and received 32.5% coverage from the protected areas, indicating that conservation gaps still existed in the existing protected-area system.