In this work, we fabricated a novel graphene film composite biosensor for microcystin-LR detection as an alternative to time-consuming, expensive, non-portable and often skills-demanding conventional methods of analysis involved in water quality monitoring and assessment. Excellent linear correlation (R2 = 0.99) of the electron-transfer resistance was achieved over a wide range of microcystin-LR (MC-LR) concentration, i.e. 0.005-10 μg/L. As-prepared graphene film composite biosensors can specifically detect MC-LR with remarkable sensitivity and detection limit (2.3 ng/L) much lower than the World Health Organization (WHO) provisional guideline limit of microcystin-LR concentration (i.e. 1 μg/L) in different water sources. Their great potential can be attributed to large active surface area of graphene film and efficient charge transfer process enabled by their high conductivity. Developed graphene film composite biosensors were also successfully applied to determination of MC-LR in several environmental water samples with high detection recovery, which offers a promising possibility of large-scale manufacture of sensor tips due to their macroscopic free-standing nature, the scalable fabrication route and easily tunable size.