A novel, facile and inexpensive solid phase extraction (SPE) method using ethylene glycol bis-mercaptoacetate modified 3-(trimethoxysilyl)-1-propanethiol grafted Fe3O4 nanoparticles coupled with spectrofluorimetric detection was proposed for determination of aflatoxin M1 (AFM1) in liquid milk samples. The method uses the advantage fluorescence enhancement by β-cyclodexterin complexation of AFM1 in 12% (v/v) acetonitrile–water and the remarkable properties of Fe3O4 nanoparticles namely high surface area and strong magnetization were utilized to achieve high enrichment factor (57) and satisfactory extraction recoveries (91–102%) using only 100mg of magnetic adsorbent. Furthermore, fast separation time of about 15min avoids many time-consuming column-passing procedures of conventional SPE. The main factors affecting extraction efficiency including pH value, desorption conditions, extraction/desorption time, sample volume, and adsorbent amount were evaluated and optimized. Under the optimal conditions, a wide linear range of 0.04–8ngmL−1 with a low detection limit of 0.015ngmL−1 was obtained. The developed method was applied for extraction and preconcentration of AFM1 in three commercially available milk samples and the results were compared with the official AOAC method.