Aflatoxin B1 (AFB1) is a very hazardous carcinogen, readily contaminating foodstuffs and traditional Chinese medicines (TCMs) that has inspired increasing health concerns due to dietary exposure. Colloidal nanocrystals have been proposed as optical labels for aptasensor assembly, but these typically require tedious multistep conjugation and suffer from unsatisfactory robustness when used for complex matrices. In the present study, we report a rapid and sensitive method for screening for trace AFB1 levels in TCMs using a label-free fluorescent aptasensor PicoGreen dye-based strategy. Using PicoGreen to selectively measure complementary double-stranded DNA, fluorescence enhancement due to dsDNA is ‘turned off’ in the presence of AFB1 due binding of aptamer target over complementary sequence. Self-assembly of a label-free fluorescent aptasensor based on AFB1 aptamer and PicoGreen dye was performed. Due to competition between the complementary sequence and AFB1 target, this rapid method was capable of highly sensitive and selective screening for AFB1 in five types of TCMs. This proposed approach had a limit of detection as low as 0.1 μg·L−1 and good linearity with a range of 0.1–10 μg·L−1 (0.1–10 ppb). Among the 20 samples tested, 6 batches were found to be contaminated with AFB1 using this method, which was confirmed using sophisticated liquid chromatography-electrospray ionization-tandem mass spectrometry/mass spectrometry analysis. The results of this study indicate the developed method has the potential to be a simple, quick, and sensitive tool for detecting AFB1 in TCMs.