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.