OBJECTIVE To investigate the clinical efficacy of three-dimensional conformal radiotherapy (3D-CRT) for locally advanced or postoperatively relapsed rectal cancer, and to examine the changes in cancer multi-biomarkers.METHODS Sixty patients with locally advanced or postoperatively relapsed rectal cancer were randomly divided into two groups after 40 Gy external radiation, namely a late-course 3D-CRT group and a conventional radiotherapy group that served as the control. There were 30 patients in each group. For patients in the 3D-CRT group, multi-biomarkers were measured before and after radiotherapy and after relapse.RESULTS Response rates in the 3D-CRT and the control groups were 86.7% (26/30) and 70% (21/30) respectively, without a significant difference (P＞0.05). The 1-, 2- and 3-year survival rates were 80%, 53.3% and 36.7% in the 3D-CRT group; in the control group the rates were 56.7%, 40% and 13.3% respectively, with a significant difference (P=0.0213). CEA, CA19-9,CA242 and FER decreased after radiotherapy in the 3D-CRT group, P＜0.01,indicating a significant difference. The values after relapse were higher than those without relapse, P＜0.01, indicating a significant difference.CONCLUSION Conventional radiotherapy with a 3D-CRT boost gives better therapeutic effect to patients with locally advanced or postoperatively locally relapsed rectal cancer. A multi-biomarker protein chip diagnosis system can be utilized as an effective tool to determine the therapeutic effect and prognosis.