Multiplexed detection of biomarkers, i.e., simultaneous detection of multiple biomarkers in a single assay, is a process of great advantages including enhanced diagnostic precision, improved diagnostic efficiency, reduced diagnostic cost, and alleviated pain of patients. A typical lateral-flow immunoassay (LFIA) is a widely used paper-based immunochromatographic test strip designed to detect a target biomarker through two common formats: sandwich assay and competitive assay. In order to obtain qualitative or quantitative results, a probe with unique optical or magnetic properties is usually employed to characterize the concentration of the target biomarker. The typical LFIA is suitable for point-of-care testing due to its simplicity, portability, cost-effectiveness, and rapid detection of a target biomarker. However, detection of a single biomarker in the typical LFIA is not favorable for high throughput analysis. Therefore, multiplexed detection of biomarkers in LFIAs has been extensively studied in recent years for high throughput analysis. To accomplish multiplexed detection of biomarkers in LFIAs, the most frequently used structure is a test strip with multiple test lines (TLs), where each TL can detect a specific biomarker. An alternative structure, i.e., a multi-channel structure with multiple test strips, where each test strip has one TL for detecting a specific biomarker, is employed for multiplexed detection of biomarkers. Sometimes, a single test strip with only one TL containing different receptors, where each detection receptor corresponds to a specific biomarker, is another structure applied for multiplexed detection of biomarkers. This paper reviews three common structures for multiplexed detection of biomarkers in LFIAs, i.e., a test strip with multiple TLs, a multi-channel structure with multiple test strips, and a test strip with a single TL. Based on the three common structures, different signal detection strategies that include colorimetric detection, fluorescence detection, surface-enhanced Raman scattering detection, and magnetic detection, along with performance and perspectives are discussed.