Aim: In this study, we aimed to evaluate in vitro the measurement accuracy of three electronic apex locators (EALs) in three different embedding media. Material and Methods: Thirty maxillary central incisors were included (N = 30). The specimens were decoronated at cement-enamel junction, and the soft tissues in root canals were removed. The samples were randomly divided into three groups (n = 10). The specimens were embedded in alginate (group A), gelatin (group B) and agar agar (group C), leaving the coronal 2 mm of teeth exposed. Electronic lengths (ELs) of root canals of samples were measured using Raypex 6 (VDW, Munich, Germany), Apex ID (SybronEndo, Glendora, USA) and Ipex 2 (NSK Inc., Kanuma, Japan). In all groups, actual length (AL) values subtracted from EL values, and EL-AL values were recorded. Results: The EL-AL values for each embedding media were classified within an error range of ± 0.5 mm and ± 1 mm, and the chi-square (χ2) test was used to compare the percentage of acceptable measurements of three EALs (α = 0.05). The measurements of Raypex 6 in agar agar medium were statistically different compared with those in alginate and gelatin media, within the error margin of ± 0.5 mm (p < 0.05). Both in alginate and gelatin media, no statistically significant difference was observed in percentages of acceptable measurements with Raypex 6, Apex ID and Ipex 2 within two ranges of error (p > 0.05). Discussion: Within the limitations of this study, alginate and gelatin can be used safely as embedding media in studies investigating the in vitro measurement accuracy of EALs.