Colorectal cancer (CRC) is a prevalent malignancy with poor prognosis and survival. As a Ca2+ binding protein, S100P plays a role in calcium-dependent signal transduction pathways that involve in diverse biological processes. Our previous studies have shown that S100P is overexpressed in CRC tissues and regulates cell growth, invasion, and metastasis in CRC. Therefore, S100P is expected to be an effective target for CRC therapy. Aptamers are short single-stranded oligonucleotides that could serve as specific and high-affinity probes to a wide range of target molecules for therapeutic purposes. In this study, we generated a novel DNA aptamer against S100P (AptS100P-1) by way of the SELEX process and high-throughput sequencing. The binding assay showed that AptS100P-1 had a high affinity for S100P protein. Further experiments indicated that AptS100P-1 is relatively stable in a cell culture system and could be used in flow cytometry analysis, dot blot assay, and fluorescence microscopy analysis to detect S100P. Moreover, AptS100P-1 was capable of binding to cells and had an inhibitory effect on CRC cell growth in vitro and in vivo. Also, AptS100P-1 inhibited the migration and epithelial-mesenchymal transition of CRC cells expressing S100P. These results indicate a novel DNA aptamer targeting S100P, which might be a potential therapeutic strategy for targeting S100P against S100P-expressing CRC.