BACKGROUND The aim of this study is to assess the value of contrast-enhanced ultrasound (CEUS) as a new non-invasive approach to locate the testicular area in which spermatogenesis is most likely to be found in non-obstructive azoospermic testes and to evaluate the accuracy of CEUS as a predictor of successful sperm retrieval. METHODS CEUS was performed in 120 nonobstructive azoospermia (NOA) patients. Microdissection testicular sperm extraction (M-TESE) was performed on the best and poorest perfusion areas selected by CEUS and on conventional areas. RESULTS In the 187 testicles that underwent M-TESE, the sperm retrieval rates (SRRs) in the best perfusion area and poorest perfusion area over the maximal longitudinal section and conventional area were 63.1, 34.7 and 47.1%. According to receiver operating characteristic (ROC) analysis, the arrival times (AT) ≤27 s, time-to-peak intensity (TTP) ≤45 s, and peak intensity (PI) ≥11 dB were the best predictors of positive sperm retrieval. The location of the best perfusion area was able to guide M-TESE to improve the success rates. CONCLUSIONS Testicle CEUS is suggested to be performed in all patients with NOA. If AT≤27 s, TTP ≤ 45 s or PI≥11 dB are found in the best perfusion area, M-TESE is strongly recommended.