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An effective fluorescent probe (mSiO2-N-CDs) was prepared by embedding N-CDs into mesoporous silica via a simple one-pot hydrothermal reaction and applied to the detection of dopamine (DA). Mesoporous silica not only provided a skeleton to prevent the aggregation of N-CDs but also a medium for the centrifugal collection of N-CDs, avoiding the need for dialysis and freeze-drying. The formation process, phase composition, morphology, and luminescence properties of the composite were studied in detail. The synthesized mSiO2-N-CDs possessed spherical morphology, a smooth surface, and a diameter of approximately 150 nm. The fluorescence results indicated that mSiO2-N-CDs emitted intense blue color fluorescence at 465 nm under the optimal excitation of 370 nm. Because the mesoporous silica effectively inhibited the self-quenching caused by the aggregation of N-CDs, the quantum yield of solid mSiO2-N-CDs powder reached 32.5%. Furthermore, the emission intensity of the solid mSiO2-N-CDs remained constant for 28 days. The good sensitivity and selectivity of mSiO2-N-CDs for DA enabled the establishment of a rapid, simple, and sensitive DA detection method. The linear range was 0–50 µM and the limit of detection was calculated to be 107 nM. This method was used for the determination of DA in urine, with recovery rates ranging between 98% and 100.8%. In addition, the sensing mechanism was characterized by fluorescence lifetime decay and UV–VIS spectral analysis. [ABSTRACT FROM AUTHOR]