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Conventional lateral flow assay (LFA) is typically performed by observing the color changes in the test lines by naked eyes, which achieves considerable commercial success and has a significant impact on the fields of food safety, environment monitoring, disease diagnosis, and other applications. However, this qualitative detection method is not very suitable for low levels of disease biomarkers’ detection. Although many nanomaterials are used as new labels for LFA, additional readers limit their application to some extent. Fortunately, a lot of work has been done for improving the sensitivity of LFA. In this review, currently reported LFA sensitivity enhancement methods with an objective evaluation are summarized, such as sample pretreatment, the change of flow rate, and label evolution, and future development direction and challenges of LFAs are discussed.