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It is recognized that cystatin C is an effective marker for monitoring the glomerular filtration rate and the clinical diagnosis of various diseases. In this study, a novel immunochromatographic method has been established to achieve quantitative detection of serum cystatin C. Unlike conventional and traditional gold immune chromatographic assays (GICAs), a uniform layer of a fluorescent film was added to the solid phase, which has been designated as the background fluorescence quenching immune chromatographic assay (bFQICA). Under the optimized conditions, there was a good correlation between the fluorescence signal ratio (F1/F2) of the background fluorescence (F1) to the fluorescence signal at the detection line (F2) for cystatin C at concentrations from 0.0 ng/mL to 100 ng/mL with a correlation coefficient of 0.9977. The detection limit was 0.69 ng/mL, and the recovery values were 87.9–105%. The differences between the intra- and interbatch precision were less than 15% in three batches. In addition, after 120 serum samples were detected, there were no significant differences in the results obtained by bFQICA and immunoturbidimetry (t = 0.963, p = 0.338 > 0.05). This work demonstrates that bFQICA is a simple, sensitive, and accurate approach for the determination of serum cystatin C, providing a new approach for the clinical diagnosis of cystatin C. [ABSTRACT FROM AUTHOR]