Early cancer diagnosis is very important for prevention or mitigation of metastasis. However, we must improve the diagnosis and assessment of cancer by an effective and efficient method. Here, we report a single-step detection method using nanoplasmonic aptamer sensor (aptasensor), targeting a vascular endothelial growth factor-165 (VEGF165), a predominant biomarker of cancer angiogenesis. Our single-step detection is accomplished by: (1) specific target recognition by an aptamer-target molecule interaction; (2) direct readouts of the target recognition. The readout is achieved by inactivation of surface plasmon enhancement of fluorescent probes preattached to the aptamers. Our aptasensor provides the appropriate sensitivity for clinical diagnostics with a wide range of linear detection from 25 pg/mL to 25 µg/mL (= from 1.25 pM to1.25 µM), high specificity for VEGF165 against PDGF-BB, osteopontin (OPN), VEGF121, and NaCl, and temporal/thermal/biological stability. In experiments with 100 % serum and saliva from clinical samples, readouts of the aptasensor and an ELISA for VEGF165 show good agreement within the limit of the ELISA kit. We envision that our developed aptasensor holds utility for point-of-care cancer prognostics by incorporating simplicity in detection, low-cost for test, and required small sample volume.