A non-enzymatic amperometric sensor using natural molybdenite (MLN) electrodeposited with methylene blue (MB) has been fabricated and characterized and its analytical performances were investigated for the determination of ascorbic acid (AA). The surface morphology of the electrode modified by electrodeposited MB was studied by use of the Advanced Mineral Identification and Characterization System (AMICS) and laser confocal high-temperature scanning microscope (LCSM). The poly(MB) and MLN immobilized sensor showed good stability, reproducibility, sensitivity, and selectivity. It exhibited a linear performance range from 3 to 1000 mu M, with a lower detection limit of 0.083 mu M (signal/noise = 3) and short response time (< 5 s). No obvious decrease in the current was observed after 20 days storage. The methodology reproducibility of this sensor was 2.6%. It showed good anti-interference ability for the potential interfering compounds. The poly(MB) film not only can enhance the electron-transfer rate but also increase the lifetime of the sensor. This study demonstrated the applicability of natural molybdenite for the fabrication of non-enzymatic electrochemical AA sensor.