Aim To identify a serum microRNA(miRNA) expression profile that can serve as a novel diagnostic biomarker for myocardial bridging (MB) detection. Methods Serum samples were taken from 100 MB patients and 50 controls. An initial screening of miRNA expression by microarray was performed using serum samples pooled from 20 MB patients and 10 controls, respectively. Differential expression was validated using hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (RT-qPCR) in individual samples. Results The microarray results demonstrated that 21 serum miRNAs were markedly different in the MB patients compared with the controls. The RT-qPCR analysis further identified a profile of four serum miRNAs (miR-92a, miR-487a, miR-29b and miR-126) as a biomarker for MB detection. The areas under the receiver operating characteristic (ROC) curve of this four-serum miRNAs were 0.998, 0.956, 0.719 and 0.986 respectively, and using the optimal cutoff value, we obtained the following sensitivity and specificity values to detect MB: 97% and 100%, 97% and 100%, 45.5% and 100%, 87.9% and 100% respectively. Conclusions We identified four serum miRNAs signature for MB diagnosis by genome-wide serum miRNA expression profiling. Expression levels of this serum miRNA may be a novel biomarker for early detection of MB in human.