Digoxin is a cardiac glycosylated steroid-like drug with a positive inotropic effect and has been widely used in treating congestive heart failure, atrial fibrillation, atrial flutter, and other heart diseases. Digoxin is also a dangerous drug, which can cause drug poisoning at a low blood drug concentration (2.73–3.9 nmol/L, i.e., 2.14–3.05 ng/mL). Therefore, the timely detection of a patient’s blood drug concentration plays a significant role in controlling blood drug concentration, reducing the occurrence of drug poisoning events, and maximizing the role of drug therapy. In this study, a DNA vector for the expression of the antidigoxin antibody Fab fragment was constructed. With the vector, Fab was expressed in E. coli and purified, and 1.2 mg of antibodies was obtained from 100 mL of culture. An immunofluorescent sensor based on the mechanism of photoinduced electron transfer was constructed by labeling additional cysteines in the heavy chain variable region and light chain variable region of the antibody Fab fragment with fluorescent dyes. The assay for digoxin with the immunosensor could be finished within 5 min with a limit of detection of 0.023 ng/mL, a detectable range of 0.023 ng/mL to 100 μg/mL, and an EC50 of 0.256 ng/mL. A new approach for the rapid detection of digoxin was developed and will contribulte to therapeutic drug monitoring.

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