DNA methylation is a critical part of epigenetics and plays a vital role in maintaining normal cell function, genetic imprinting, and human tumorigenesis. Thus, it is important to develop a sensitive method for the determination of DNA methyltransferase (MTase) activity. Here, we present a simple and sensitive method based on single molecule fluorescence correlation spectroscopy (FCS) and polystyrene polymer dots (PS Pdots) for the quantitative detection of DNA adenine methylation (Dam) MTase activity and its inhibitor screening in homogeneous solution without separation. Its principle is based on the measurement of the characteristic diffusion time (τ_D) of unmethylated and methylated DNA-fluorescent probes by FCS. A hairpin DNA probe including the 5′-GATC-3′ sequence is used by doubly labelling fluorophore Alexa Fluor 488 (Alexa 488) and biotin at the 5′- and 3′-terminus, respectively. Dam MTase catalyzed the methylation of the sequence of 5′-GATC-3′, and DpnI cleaved the sequence of 5′-G-Am-TC-3′. Streptavidin conjugated PS Pdots were used to react with DNA probes without methylation to further increase the difference in τ_D values between methylated and unmethylated DNA-Alexa 488 probes. We used the FCS method to measure the τ_D values of DNA-Alexa 488 probes and further obtained the activity of Dam MTase. It is found that the τ_D value of the methylated DNA probe is negatively correlated with the logarithm of Dam MTase concentration in the range from 0.025 U mL⁻¹ to 3 U mL⁻¹. The detection limit is as low as 0.025 U mL⁻¹. Furthermore, we evaluated the inhibition effect of drug-related DNA methylation and the half-maximal inhibitory concentration (IC₅₀) value is consistent with a previous study. The results demonstrated that our proposed method will become a promising platform for the determination of Dam MTase activity and inhibitor screening.