The invention relates to the technical field of biosensors, and particularly relates to a biosensor for detecting exosome based on a nucleic acid aptamer. The biosensor comprises an aptamer PTK-7 Apt,CD63 Apt, a connector, a hairpin probe H1, a hairpin probe H2 and a CCRF-CEM. The invention also relates to a production method and an application thereof. Based on specific recognition of the nucleic acid aptamer and a target object, through connection of the connector, two chains of PTK-7 Apt and CD63 Apt are adjacent to each other to form a trigger, and the trigger further opens H1 and the H1opens H2 to trigger an HCR reaction so that amplification of chemiluminescence intensity signals is realized, and the aptamer biosensor is constructed. The sensor has advantages of a high detection speed, simplicity in operation, a low price, a low detection limit, high specificity and the like.