The aim of the present study was to evaluate the agreements between the root canal length estimations using cone-beam computed tomography (CBCT) at different slice thicknesses, dedicated software, or an electronic apex locator (EAL) and the actual lengths (AL). In total, 111 extracted human molars with 302 root canals were chosen. Teeth were scanned using a CBCT device at a voxel size of 0.075 mm. Root canal lengths were estimated using CBCT software at different slice thicknesses (0.6, 1.2, and 2.4 mm) and dedicated software for proposed or operator lengths. The endodontic access cavities were created, and root canal lengths were estimated with an EAL for electronic length (EL) and a ruler for AL. Data were tested using paired t-tests and Bland–Altman plots to detect the differences between the methods in length estimation at a significance of 0.05. The accuracy in the range of ± 0.5 mm was 100% for the EAL. There was an agreement between the EL and CBCT at a slice thickness of 1.2 mm (p = 0.349). CBCT at the smallest slice thickness estimation was not the best modality in agreement with the AL. The EAL was an accurate and reliable method for root canal length measurement.