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Creatinine measurement in blood and urine is an important diagnostic test for assessing kidney health. In this study, a molecularly imprinted polymer was obtained by incorporating fluorescent nanodiamond into a creatinine-imprinted polyacrylamide hydrogel. The quenching of peak nanodiamond fluorescence was significantly higher in the creatinine-imprinted polymer compared to the non-imprinted polymer, indicative of higher creatinine affinity in the imprinted polymer. Fourier transform infrared spectroscopy and microscopic imaging was used to investigate the nature of chemical bonding and distribution of nanodiamonds inside the hydrogel network. Nanodiamonds bind strongly to the hydrogel network, but as aggregates with average particle diameter of 3.4 ± 1.8 µm and 3.1 ± 1.9 µm for the non-imprinted and molecularly imprinted polymer, respectively. Nanodiamond fluorescence from nitrogen-vacancy color centers (NV− and NV0) was also used to detect creatinine based on nanodiamond-creatinine surface charge interaction. Results show a 15% decrease of NV−/NV0 emission ratio for the creatinine-imprinted polymer compared to the non-imprinted polymer, and are explained in terms of changes in the near-surface band structure of diamond with addition of creatinine. With further improvement of sensor design to better disperse nanodiamond within the hydrogel, fluorescent sensing from nitrogen-vacancy centers is expected to yield higher sensitivity with a longer range (Coulombic) interaction to imprinted sites than that for a sensor based on acceptor/donor resonance energy transfer.