Background. To explore the application of serum proteomic patterns for the preoperative detection of regional lymph node involvement of colorectal cancer (CRC). Methods. Serum samples were applied to immobilized metal affinity capture ProteinChip to generate mass spectra by Surface-Enhanced Laser Desorption/ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). Proteomic spectra of serum samples from 70 node-positive CRC patients and 75 age- and gender-matched node-negative CRC patients were employed as a training set, and a classification tree was generated by using Biomarker Pattern Software package. The validity of the classification tree was then challenged with a blind test set including another 65 CRC patients. Results. The software identified an average of 46 mass peaks/spectrum and 5 of the identified peaks at m/z 3,104, 3,781, 5,867, 7,970, and 9,290 were used to construct the classification tree. The classification tree separated effectively node-positive CRC patients from node-negative CRC patients, achieving a sensitivity of 94.29% and a specificity of 100.00%. The blind test challenged the model independently with a sensitivity of 91.43% a specificity of 96.67%. Conclusions. The results indicate that SELDI-TOF-MS can correctly distinguish node-positive CRC patients from node-negative ones and show great potential for preoperative screening for regional lymph node involvement of CRC.