Biochemical sensors have found widespread applications in the fields of health and environment. As the number of biochemical sensors continues to increase, their energy supply has emerged as a challenge. Self-powered biochemical sensors based on triboelectric generators (TENGs) offer a promising solution to this challenge. However, current self-powered sensors for in situ detection of liquid samples either suffer from low output signals, resulting in insufficient sensitivity, or require relatively large sample volumes. To address these challenges, this work introduces a TENG with charge separation capability by incorporating electrodes that can directly contact the solution at both ends of a fluidic channel. Unlike traditional devices, this device utilizes the reciprocating motion of a single droplet within the device to achieve charge accumulation in both the electrodes and the liquid sample. By utilizing the characteristic that biochemical substances contained within the droplet affect its charge storage capacity, the concentration of these biochemical substances in the droplet is reflected by the value of the output voltage when it reaches a stable state. The device functions as a triboelectric charge-separable probe and demonstrates responsiveness to solution pH, salt concentration, and the concentration of nanoparticles or Escherichia coli, showcasing its potential as a biochemical sensor.