DNA methyltransferases1 (DNMT1), a dominant enzyme responsible for maintaining the genetic stability of DNA methylation, is closely related to the occurrence and development of a variety of tumors. However, till now, most of the studies on DNA methyltransferases (DNMTs) detection have focused on prokaryote methyltransferases and been limited to laboratory studies. Therefore, a fluorescence immunoassay (FLISA) for the high sensitivity and high throughput detection of DNMT1 level in human serum samples was established to provide new ideas for early cancer diagnosis and clinical cancer therapy. The functional carboxyl Fe3O4 magnetic beads were used as solid phase carriers. 1-chloride-3-dimethylamino-propyl-3-ethylcarbodiimide hydrochloric acid (EDC) and sulfo-N-hydroxysuccinimide (Sulfo-NHS) were used as coupling agents to prepare immune-magnetic capture probe Fe3O4@ McAbDNMT1 (monoclonal antibody DNMT1). After that, the immune-fluorescent detective probe 5-TAMRA@PcAbDNMT1（5-carboxytetramethylrhodamine@polyclonal antibody DNMT1）was also made. Their structure and activity were characterized by infrared absorption, UV-Vis spectra, Zeta potential and immunocompetence. Then, the content of DNMT1 in human serum samples was detected based on the double-anti-sandwich method by FLISA with high sensitivity. Finally, methodology evaluation and comparison were conducted. The results showed that the probes conjugated successfully and maintained the immunocompetence of the original antibody. The linear equation was y=222.046+48.323x, the linear range was 0.05～80 ng·mL-1, and the correlation coefficient was 0.991 4, the detection limit was 0.005 ng·mL-1, the intra-and inter-panel RSD was 4.7%～8.8% and 1.6%～10.0% (n=6), respectively, intra- and inter-panel recoveries were 91.3%～102.4% and 88.0%～98.8% (n=6), respectively. The cross-reactivity rates with other two DNA methyltransferase 3a/3b were lower, and the specificity of FLISA was well. Compared with ELISA and MELISA, FLISA has the lowest detection limit and shortest analysis time. Compared with ELISA kits, the result displayed a high correlation between two methods, and the difference of them was not statistically significant (p＞0.05). The result suggests that the FLISA system would be used to detect multiple samples at the same time for the rapid analysis of DNMT1 in human serum samples.