A simple and rapid detection strategy for vitamin B 12 (VB 12 ) was established based on label-free silicon quantum dots (SiQDs); the detection mechanism was additionally investigated. SiQDs were synthesized using a one-step microwave method, and their fluorescence was stronger than that synthesized using the hydrothermal method.SiQDs fluorescence was quenched using VB 12 due to the inner filter effect (IFE), which was demonstrated using ultraviolet (UV) absorption spectra, fluorescence lifetime, transmission electron microscopy and zeta potential analysis. Subsequently, quercetin (Que) and doxorubicin (Dox) with absorption peaks that overlapped the excitation or emission peaks of SiQDs respectively were used as control groups to investigate the quenching mechanism. Results showed that quenching efficiency was related to the level of overlap between the adsorption peak of the quencher and the excitation or emission peaks of SiQDs. A greater level of overlap caused a higher quenching efficiency. Therefore, the sensitive quenching of VB 12 for SiQDs was due to the synergistic effect of the synchronous overlap between the absorption peak of VB 12 with the excitation and emission peaks of SiQDs. Fluorescence quenching efficiency increased linearly in the 0.5 to 16.0 μmol·L −1 VB 12 concentration range, and the detection limit was 158 nmol·L −1 . In addition, SiQDs were applied to determine VB 12 in tablets and human urine samples with satisfactory recoveries ranging from 97.7 to 101.1%. KEYWORDS fluorescence method, inner filter effect, quenching mechanism, silicon quantum dots, vitamin B 12