Conventional in vitro circulating tumor cell (CTC) detection methods are always limited by the blood sample volume because of the extremely low abundance of CTCs among the large number of hematologic cells. The aim of this study was to overcome this limitation by designing and constructing an in vitro CTC capture assay. We blended poly(sulfobetaine methacrylate) (PSBMA) and poly(acrylic acid) (PAA) into nylon-6 through electrospinning to generate a fibrous mat-based capture assay of CTCs. The contents of nylon-6, PSBMA, and PAA in the electrospun triple-blend fibrous mats (ETBFMs) were optimized to avoid degradation and to balance between the non-biofouling behavior and the antibody immobilizing efficiency. In addition, we examined the capture ability of CTCs for clinical diagnoses of colorectal cancer, in comparison with the results identified through pathological analyses of biopsies from colonoscopies. For nine individuals with stage II, III, and IV colorectal cancer, our CTC detection with ETBFMs provided complete positive expression. Two of four individuals were diagnosed to possess stage I colorectal cancer. Two of seven individuals without colorectal tumor, as identified through pathological analyses of biopsies, exhibited positive expression of CTCs. These positive results suggest that such ETBFMs are promising materials for in vitro CTC capture assays.