<p>A novel self-calibrating triple-emission fluorescent probe (CDs/BSA-SQ-Cy7) was engineered through the strategic integration of blue-emitting carbon dots (CDs) with covalently assembled near-infrared fluorescent dyes (BSA-SQ and BSA-SQ-Cy7) for accurate in vitro pepsin detection. To validate the enhanced performance of the tri-emissive design, a dual-emission control probe (CDs/BSA-SQ) was developed. BSA-SQ-Cy7 undergoes hydrolysis upon interaction with pepsin, resulting in concentration-dependent quenching of the red fluorescence, where the blue CDs serve as a reference emission during the process. The CDs/BSA-SQ-Cy7 probe demonstrated a robust linear correlation with pepsin concentrations ranging from 0.018 to 30&#xa0;μg/mL (determined by the lowest detection limit). The detection limits were found to be 0.182&#xa0;μg/mL (<i>I</i>₄₉₅/<i>I</i>₆₅₉), 0.033&#xa0;μg/mL (<i>I</i>₄₉₅/<i>I</i>₈₀₈), and 0.018&#xa0;μg/mL&#xa0;((<i>I</i>₄₉₅-<i>I</i>₈₀₈)/<i>I</i>₆₅₉), respectively. Additionally, the probe displayed excellent stability and reproducibility over a 72-h period, and its three-channel emission signal ratios can be utilized for self-calibration, thereby improving assay accuracy. This self-calibrated tri-emission probe is distinguished by its stability, reproducibility and high selectivity, offering an effective method for the accurate in vitro detection of pepsin.</p> Graphical Abstract <p></p>