Rapid and sensitive analysis of proteins in complex biological environments is crucial for the screening and defense against infectious diseases. Here, we show that the lateral flow immunoassay strip based on confocal Raman imaging can achieve immune analysis at pM and ∼ 104 cfu/mL molecular level for the rapid detection of COVID-19 virus and bacteria. Fluorescent dyes of Alexa 647 were used as Raman markers in the Raman silent region of 1800 cm−1 and 2800 cm−1, and colloidal gold nanospheres were used to enhance the Raman signal. Raman imaging was performed with our self-developed confocal Raman microscopy for COVID-19 and Escherichia coli O157: H7 on lateral flow immunoassay strip. Compared to traditional colloidal gold test strips, the sensitivity of this technology has been significantly improved. This work will promote the widespread application of surface enhanced Raman detection for bacteria and virus, which is of great significance for in vitro screening and disease diagnosis.