Abstract Background To overcome the limitations regarding two dimensional (2D) greyscale (GS) transrectal ultrasound (TRUS)-guided biopsy in prostate cancer (PCa) detection and tissue packaging in biopsy processing, there is an ongoing focus on new imaging and pathology techniques. A three-dimensional (3D) model of the prostate with biopsy needle guidance can be generate by the Navigo™ workstation (UC-care, Israel). The SmartBX™ system (UC-care, Israel) provides a prostate biopsy core preembedding method. The aim of this study was to compare cancer detection rates between the 3D TRUS-guidance and preembedding method with conventional 2D GS TRUS-guidance among patients undergoing prostate biopsies. Methods We retrospectively analyzed the records of all patients who underwent prostate biopsies for PCa detection at our institution from 2007 to 2016. The cohort was divided into a 2D GS TRUS-guidance cohort (from 2007 to 2013, n = 1149) and a 3D GS TRUS-guidance with preembedding cohort (from 2013 to 2016, n = 469). Effect of 3D GS TRUS-guidance with preembedding on detection rate of PCa and clinically significant PCa (Gleason score ≥ 7 or > 2 biopsy cores with a Gleason score 6) was compared to 2D GS TRUS-guidance using regression models. Results Detection rate of PCa and clinically significant PCa was 39.0 and 24.9% in the 3D GS TRUS cohort compared to 33.5 and 19.0% in the 2D GS TRUS cohort, respectively. On multivariate regression analysis the use of 3D GS TRUS-guidance with preembedding was associated with a significant increase in detection rate of PCa (aOR = 1.33; 95% CI: 1.03–1.72) and clinically significant PCa (aOR = 1.47; 95% CI: 1.09–1.98). Conclusion Our results suggest that 3D GS TRUS-guidance with biopsy core preembedding improves PCa and clinically significant PCa detection compared to 2D GS TRUS-guidance. Additional studies are needed to justify the application of these systems in clinical practice.