Metastasis, the capability of tumor cells to spread and grow at distant sites, is the main factor in cancer mortality. Because metastasis in sentinel lymph nodes suggests the original spread of tumors from a primary site, the detection of lymph node involvement with cancer serves as an important prognostic and treatment parameter. Here we have developed a panel of DNA aptamers specifically binding to colon cancer cells SW620 derived from metastatic site-lymph node, with high affinity after 14 rounds of selection by the cell-SELEX method. The selected aptamers were subjected to flow cytometry to evaluate their binding affinity. Aptamer XL-33 with the best binding affinity (0.7 nM) and its truncated sequence XL-33-1 with 45 nt showed excellent selectivity for the recognition of target cells SW620. The binding entity of the selected aptamer has been preliminarily determined as a membrane protein on the cell surface. Tissue imaging results showed that XL-33-1 was highly specific to the metastatic tumor tissue or lymph node tissue with corresponding cancer metastasis, and displayed an 81.7% detection rate against colon cancer tissue with metastasis in regional lymph nodes, as well as a 72.4% positive rate against lymph node tissue with colon cancer metastasis. These results suggest that XL-33-1 holds great potential to become a molecular imaging agent for early detection of lymph node tissue with colon cancer metastasis. More importantly, this study clearly demonstrates that metastatic-cell-based SELEX can be used to generate DNA ligands specifically recognizing metastatic cancer cells, which is of great significance for metastatic cancer diagnosis and treatment.