In this paper, a multifunctional microfluidic chip integrated with a centrifugal separation zone, aqueous two-phase system (ATPS) mixing zone and enrichment detection zone was proposed and fabricated. An automatic and efficient separation and quantitative analysis method for vascular endothelial growth factor 165 (VEGF165) in whole blood samples was established with the designed microfluidic chip. A blood sample was divided into blood cells and plasma in the centrifugation zone. In the ATPS mixing zone, plasma was mixed with PEG/KH2PO4 aqueous two-phase solution containing Apt-Au NP nanoprobes. In the enrichment detection zone, the mixture was separated on CN140 modified with a ZnO NP-anti VEGF165 nanostructure. The VEGF165 captured by Apt-Au NPs was distributed in the PEG phase, concentrated at the front of CN140 and combined with anti-VEGF165 to form a sandwich structure. The sensitive detection of VEGF165 was achieved through fluorescence resonance energy transfer between rhodamine B and Au NPs on the nanoprobe. Under the optimized rotation program, capillary and centrifugal forces propelled the fluid in the whole process of pretreatment and detection. The detection linear range was between 1 pg mL-1 and 50 ng mL-1, the detection limit of VEGF165 in blood was 0.22 pg mL-1 and the enrichment efficiency was 983. It was illustrated that a convenient and reliable way for detection of tumor markers based on the multifunctional microfluidic chip was provided and it has a potential value for early screening and prognosis of clinical cancer.