The invention discloses a silicon nano-sphere and nucleic acid aptamer-based bifluorescence probe as well as a manufacturing method thereof and application thereof. The manufacturing method for the probe comprises the following steps: modifying two DNA single chains complementary with nucleic acid aptamer base sequences of different kinds of target analyte on the surface of a silicon nano-sphere, and modifying two different fluorescence groups at ends 5' of two nucleic acid aptamer chains; mixing and incubating the two fluorescence groups, thereby obtaining the bifluorescence probe-based on the base complementary pairing principle. When the probe and the target analyte coexist, the target analyte will be competitively bonded with the nucleic acid aptamers, so that the nucleic acid aptamers are separated from the complementary chains thereof and drop from the probe. After a solution is centrifuged at high speed, liquid supernatant is collected and a fluorescence intensity value is measured; and concentration of each target analyte is calculated according to the fluorescence intensity values at two maximal wavelengths. The bifluorescence probe can realize simultaneously distinguishing and detecting the two kinds of target analyte in a complex sample; and moreover, operations are simple and convenient, speed is high and sensitivity is high.