Graphene-like new generation of two dimensional (2D) nanomaterials, in particular the family of transition metal dichalcogenides (TMDCs) have been attracting a wide range of research interest. Among the family of TMDCs, molybdenum disulfide (MoS2) has been the most used one for biosensor applications. In this work, we detail the preparation of MoS2/chitosan composite modified pencil graphite electrode (MoS2/Chitosan/PGE) and its use as glucose biosensor after the modified electrode were immobilized with glucose oxidase (GOx)-gelatin (MoS2/Chitosan/GOx-Gelatin/PGE). This electrode was compared with the unmodified electrode in terms of electrochemical performance. Superior electrochemical responses were achieved including good electroactivity and sensitive glucose biosensing. For the MoS2/Chitosan/GOx-Gelatin modified PGE, a linear glucose concentration range was obtained from 10 mu M to 800 mu M with the equation of y = 0.0008x + 0.0547 (R2 = 0.9992) and a limit of detection (LOD) value of 3.18 mu M. The relative standard deviation (RSD%) was calculated as 3.77% at 200 mu M glucose (n = 5). The biosensor was tested in the presence of dopamine and ascorbic acid and our results presented the high selectivity of this novel MoS2/Chitosan/GOx-Gelatin modified PGE as a glucose biosensor.