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Objective: The aim of this in vitro study was to compare fluorescence-aided caries excavation with conventional excavation based on the Martens and Vickers hardness of dentin at the cavity floor after caries removal. Materials and methods: In total, 20 extracted human teeth with dentin caries were bisected through the lesion center into two halves, which were assigned to either the fluorescence-aided caries excavation group or the conventional excavation group. After the treatment, embedding, mounting, and polishing, a line of indentations from the dental pulp across the sound dentin to the cavity floor was made on each sample. The data were compared with Student's t and Mann-Whitney U tests. Results: The calculated Vickers hardness of the sound dentin was 57 ± 10 kg/mm in the fluorescence-aided caries excavation group and 59 ± 8 kg/mm in the conventional excavation group, which is consistent with the previous studies. The absolute and relative Martens hardness measurements of the cavity floor were 224 ± 93 N/mm and 46 ± 17 %, respectively, in the fluorescence-aided caries excavation group and 412 ± 75 N/mm and 81 ± 14 %, respectively, in the conventional excavation group. Based on either the Martens or Vickers hardness, both the absolute and relative microhardness measurements of the cavity floor after fluorescence-aided caries excavation were significantly lower than the values obtained by conventional excavation. Conclusion: Fluorescence-aided caries excavation showed the tissue-preserving property and was more conservative than the conventional excavation in this in vitro study. [ABSTRACT FROM AUTHOR]