Based on the mesoporous silica nanoparticles (MSN), a novel, simple and label-free aptamer biosensor was designed for the detection of aflatoxin B (AFB). Here, the aptamers were used as molecular recognition probes and "gated molecules" while Rh6G was loaded into the interior of the particles as the signal probe. In the absence of AFB, the "gate" was closed to prevent the leakage of the signal probe because of the immobilization of aptamers on the surface of MSN-NH. With the presence of AFB, the "gate" could be opened to release the signal probe for the specifical binding of aptamers to AFB. Our results showed that the fluorescence intensity was positively correlated with the concentration of AFB (0.5-50 ng mL), with the detection limit as low as 0.13 ng mL. What's more, this design provides a new approach for rapid, sensitive and selective detection based on aptamers and it could be applied to numerous other analytes if appropriate aptamers are available.