Due to the advanced fluorescence property of N-carbon quantum dots (N-CQDs), a new method to detect pathogenic fungi by newly synthesized cornstalk N-CQDs modified with water-soluble amphotericin B (N-CQDs@AmpB) was developed. Specifically, N-CQDs with blue fluorescence were initially synthesized according to a previous report and modified with amphotericin B on their surfaces. Subsequently, the as-prepared N-CQDs@AmpB was used to detect Candida albicans, exhibiting a linear range of 2.60 × 105 to 1.99 × 108 cfu/mL and a detection limit of 1124 cfu/mL. Compared with other common methods, the method largely shortened the detection time and enabled the process to be performed with minimal interference from complex samples such as beef sausage. The high cost of water-soluble amphotericin B may hamper the large-scale application of the new detection method using N-CQDs@AmpB. Thus, alcohol-soluble amphotericin B was used in subsequent experiments, confirming its potential to broaden avenues for the detection of fungi.