Objective To investigate the clinical application value of an immunochromatographic test strip for Norovirus utilizing sensitivity-enhanced antibodies derived from protein sequence alignment methods. Methods A retrospective analysis was conducted on a cohort of 150 suspected Norovirus-infected patients serving as study subjects. All patients underwent routine examinations and were tested for Norovirus antigens by an optimized lateral flow immunoassay. Clinical data of all patients were reviewed to validate the test results. The diagnostic performance of this optimized detection method was evaluated in terms of detection rate, reproducibility, concordance rate, resistance to interference, and cross-reactivity. Results The optimized antigen test strip demonstrated significantly higher accuracy than conventional rapid antigen tests (P < 0.05). Through assessment, it was found that several key factors in Norovirus antigen detection, such as the timing of sample collection, collection method, and preservation techniques, had substantial impacts on the test outcomes. By controlling and managing these factors, the accuracy and reliability of Norovirus antigen detection could be substantially improved. Conclusion Clinical application of the Norovirus test strip utilizing sensitivity-enhanced antibodies derived from protein sequence alignment methods presents high value, as it can refine detection methodologies, enhance both accuracy and reliability, and thereby reducing misdiagnosis and missed diagnoses.