Objective: To compare the consistency of the biological widths measured by using cone-beam CT (CBCT) and periodontal probe in patients with two different gingival biotypes. Methods: Totally 27 patients [13 males, 14 females, (37.6±13.7) years old], who planned to receive the crown lengthening surgery, were recruited under the inclusion and exclusion criteria in Department of Periodontology, School of Stomatology, The Fourth Military Medical University during November 2017 to June 2018. A total of 40 teeth (14 front teeth, 26 posterior teeth) were involved in this study. The patients were divided into two groups according to their gingival biotypes: thin gingival biotype [5 males, 8 females, (40.2±15.0) years old, 21 teeth] and thick gingival biotype [8 males, 6 females, (35.1±11.9) years old, 19 teeth]. All the teeth were checked before crown lengthening procedures by using CBCT, and the biological widths and sulcus depths were measured during the surgery by using periodontal probes (Hu-Friedy, U S A). The data were recorded and statistically analyzed. Results: There were no significant differences of the biological widths between the two measuring methods amongst all of the 40 teeth [periodonial probe: (1.64±0.26) mm; CBCT: (1.69±0.20) mm], amongst 21 thin gingival biotype teeth [periodontal probe: (1.49±0.19) mm; CBCT: (1.57±0.12) mm] and amongst 19 thick gingival biotype teeth [periodontal probe: (1.80±0.21) mm; CBCT: (1.87±0.18) mm] (P>0.05). There were no significant differences of the biological widths [anterior teeth: (1.59±0.15) mm, posterior teeth: (1.67±0.29) mm, P=0.42] and of the sulcus depths [anterior teeth: (2.00±0.28) mm, posterior teeth: (2.11±0.43) mm, P=0.44] between anterior teeth and posterior teeth. The difference of biological widths, measured by two methods respectively, between thin and thick gingival biotype groups was statistically significant (P<0.01). There were significant differences of the sulcus depths, measured by the periodontal probes, between the thin [(1.93±0.28) mm] and thick [(2.24±0.41) mm] gingival biotype groups (P<0.01). Conclusions The biological widths measured by CBCT is consistent with those measured by using periodontal probes. The biological widths and the depths of the sulcus of thin and thick gingival biotypes are different.