Objective To evaluate the effect of chemotherapy on detection of colorectal cancer autoantibodies by phage display peptide method. Methods The five-phage peptide clones (No.95, No.149, No.174, No.396, and No.1009) with high discriminatory ability of colorectal cancer patients were selected as the study subjects. We compared the reactivity of autoantibodies against each of the five-phage peptide clones among 20 colorectal cancer patients receiving chemotherapy, 40 colorectal cancer patients receiving no chemotherapy, and 40 healthy controls by enzyme linked immunosorbent assay (ELISA). Results The seroreactivity of No.95 clone was significantly lower in the patients with chemotherapy than those without chemotherapy (P<0.05), and was similar between normal control group and patients with chemotherapy (P=0.074). For the seroreactivity of the other four clones (No.149, No.174, No.396, No.1009), there were significant differences between patients with chemotherapy and normal controls (all P<0.01). Conclusion The detection ability of No.95 clones for colorectal cancer autoantibodies is greatly influenced by chemotherapy, while chemotherapy shows no notable influence on the reactivity of other four-phage peptide clones.