Since the exosomal protein level is related to many diseases, sensitive detection of exosomal protein is highly desirable. Here, we describe a polymer-sorted high-purity semiconducting carbon nanotubes (CNTs) films-based field-effect transistor (FET) biosensor for ultrasensitive and label-free detection of MUC1, a transmembrane protein highly expressed in breast cancer exosomes. Polymer-sorted semiconducting CNTs hold advantages including high purity (>99%), high CNT concentration, and short processing time (<1 h), but they are difficult to be stably functionalized with biomolecules because of lacking hanging bonds on their surface. To solve this issue, poly-lysine (PLL) was employed to modify the CNT films after they were deposited on the sensing channel surface of the fabricated FET chip. To specifically recognize the exosomal protein, sulfhydryl aptamer probes were immobilized on the gold nanoparticles (AuNPs) surface that was assembled on PLL substrate. The aptamer-modified CNT FET was capable of sensitively and selectively detecting exosomal MUC1 as high as 0.34 fg/mL. Moreover, the CNT FET biosensor was able to recognize breast cancer patients from healthy individuals by comparing the expression level of exosomal MUC1. The developed CNT FET biosensor is expected to be a novel assay for early diagnosis of cancer.