Highlights • Nitrogen-doped carbon dots with high quantum yield were prepared by pyrolyzing folic acid. • The prepared MnO 2 nanosheets were stabilized in mildly acidic environment. • N-CDs-MnO 2 nanocomposites were used for sensitive and selective detection of acid phosphatase. • l -Ascorbic acid-2-phosphate could be hydrolyzed by acid phosphatase to produce AA. Abstract In this work, a convenient and effective fluorescent “Switch-On” nanosensor was developed for the sensitive and selective detection of acid phosphatase (ACP) activity based on nitrogen-doped carbon dots (N-CDs)-manganese dioxide (MnO 2) nanocomposites. Due to the formation of N-CDs-MnO 2 nanocomposites, the fluorescence of N-CDs could be greatly quenched by MnO 2 nanosheets through fluorescence resonance energy transfer (FRET). Upon the introduction of L-ascorbic acid (AA), MnO 2 nanosheets could be reduced to Mn2+ ions, which resulted in sufficient recovery of fluorescent signal. Thus, the ACP can be monitored based on the enzymatic hydrolysis of l -ascorbic acid-2-phosphate (AAP) by ACP to generate AA. The quantitative determination of ACP activity in the range of 5–40 U L−1 with the detection limit of 0.1 U L−1 can be achieved in this sensing system. Endowed with high sensitivity and selectivity, the developed nanosensor is capable of monitoring ACP level in human serum samples with satisfactory results, indicating its great potential to be used in clinical diagnosis. [ABSTRACT FROM AUTHOR]