Amyloid-β peptide (Aβ) aggregation, one of the major hallmarks of Alzheimer’s disease (AD), has been commonly recognized as the critical culprit in AD etiology. However, the exact mechanism of Aβ aggregation in AD is still not fully understood. Moreover, development of methods capable of probing the aggregation without any perturbations has yet to be achieved. To address this challenge, we herein report a label-free fluorescent probe (BTS) for dynamic in situ visualization of Aβ40 aggregation. BTS is able to bind to all forms of Aβ40 with high binding affinity, accompanied with similar fluorescence enhancement. Intriguingly, BTS does not affect Aβ40 aggregation, probably due to its unique binding mode targeting the N-terminus of Aβ40 as the irresponsible region for aggregation. Importantly, with the prominent fluorescence performances including high selectivity and sensitivity (168 nM) as well as large Stokes shift (185 nm), BTS can veritably monitor entire Aβ aggregation process through fluorescence imaging. The probe would provide a promising tool for exploring Aβ aggregation-related pathogenesis and treatment of AD.