To evaluate the effects of different treatment time of 5.25% Sodium hypochlorite (NaOCl) on the microshear bond strength (μSBS), attenuated total reflection Fourier transform infrared (ATR-FTIR) and etching pattern in mild and moderate fluorotic enamel. Forty-eight fluorotic molars were divided into two groups: mild and moderate fluorotic enamel which were classified by a Thylstrup and Fejerskov index (TFI). Based on the application time (0s, 60s, 120s, 180s) of 5.25% NaOCl, each group was sectioned into four parts. Then the etched enamel was bonded with resin and tested to acquire μSBS. The statistical method was two-way ANOVA and Least Significant Difference (LSD) test at α = 0.05. Besides, fracture modes were observed under a stereo microscope. SEM was used to evaluated the enamel-etching pattern and organic content on the fluorotic enamel surface were investigated by ATR-FTIR. Duration of 5.25% NaOCl at 60s or 120s significantly increased the μSBS of fluorotic enamel compared to 0s (p<0.05). Fracture modes indicated that dominating failures were set in the bonding interface but whose proportion decreased when 5.25% NaOCl was applied. The enamel-etching pattern in 180s was deepest under SEM. Spectra of enamel samples manifested an obvious and gradual removal of its organic phase after duration of NaOCl increased. The maximal μSBS is acquired by using 5.25% NaOCl at 60s for mild fluorotic enamel but 120s for the moderate. The prolonged application time of 5.25% NaOCl prior to phosphoric acid etching improves enamel-etching pattern. Treatment of 5.25% NaOCl decreases proteins on the fluorotic enamel surface.