N-glycolylneuraminic acid (Neu5Gc) is a type of sialic acid that is not typically produced in healthy humans but detective in some visceral cancer cells. As a new carcinoma biomarker, the level change in the serum and urine from the patient could potentially have the relation to the disease progression. So the measurement of the Neu5Gc will help to take a better response to therapeutic schedule for the sufferers. A sensitive and rapid aptamer-nanoparticle immunochromatographic strip for the visual detection of Neu5Gc was developed. The assay is based on the competitive reaction of binding the DNA aptamer targeting the candidate molecule selected by SELEX between Neu5Gc and complementary DNA. The sensing results indicated that the aptamer-based strip was sufficiently sensitive to detect Neu5Gc. The visual limit of detection (LOD) for semi-quantitative detection was 30 ng/mL under the optimal conditions and a quantitative detection limit of 5.38 ng/mL could be obtained using a scanning strip reader. The average recovery of the spiked cancer cell samples was 88.86%, with a coefficient of variation (CV) of 5.27%. The detection could be performed in less than 15 min using a simple procedure without any complicated equipment, demonstrating that this aptamer-nanoparticle biosensor strip has great potential for use to Neu5Gc-related cancer diagnosis.