Acute myocardial infarction (AMI) is recognized as one of the deadliest cardiovascular diseases in the world, causing serious public health problems worldwide. Myoglobin (Mb) is an important biomarker for AMI. Therefore, the accurate detection of Mb is crucial for the early detection and clinical diagnosis of AMI. In this study, a novel imidazole Compound C and novel Fe3O4@4-VP microspheres were prepared and used to construct an unlabeled fluorescence aptasensor for Mb detection. Compound C can bind to the aptamer of Mb (Mb-Apt) through electrostatic attraction to form a C/Mb-Apt complex and produce the aggregation-induced emission enhancement effect. Then, Mb was added to the C/Mb-Apt solution. Mb could combine with C/Mb-Apt to form a C/Mb-Apt/Mb complex. Subsequently, Fe3O4@4-VP was added to the above mixture. The bases of Mb-Apt already combined with Mb, and thus, C/Mb-Apt/Mb would no longer interact with the pyridine rings on the surface of Fe3O4@4-VP through π-π stacking. After magnetic separation, the C/Mb-Apt/Mb complex remained in the supernate. The supernate exhibited strong fluorescence and its fluorescent intensity was proportional to the level of Mb. Experimental results showed an excellent linear relationship between the fluorescent intensity of the supernate and the level of Mb within the range of 0.167–20 ng/mL, with a detection limit reaching as low as 0.05 ng/mL (S/N = 3). In addition, the aptasensor exhibited high specificity and could be used for detecting Mb in human serum.