We developed a highly specific sensing platform for the sequential detection of two analytes in a homogenous solution by using colorimetric and fluorescence methods. This design used aptamer units [thiolated aptamer I (Apt I) for platelet-derived growth factor BB (PDGF-BB); fluorescein amidite (FAM)-labeled aptamer II (Apt II) for adenosine]. A bifunctional aptasensor was prepared by the self-assembly of thiolated Apt I on gold nanoparticles (AuNPs) and partial hybridization between Apt I and Apt II. The fabricated bifunctional aptasensor was used to detect two targets (PDGF-BB and adenosine) sequentially and selectively in a homogenous solution. In the presence of target molecules PDGF-BB, the color and absorbance of the aptamer-modified AuNPs changed because of AuNP aggregation. The increase of fluorescence intensity of FAM at 520 nm was used to analyze adenosine in the test system. The proposed aptasensor had low detection limit, high sensitivity, and bifunctional recognition and was successfully used for real serum samples. It also highlights promising applications for other aptamers and a broad range of other analytes.