Glyphosate (GLY) is a broad-spectrum herbicide and is the most used pesticide worldwide. This vast usage has raised strong interest in the ecotoxicological impacts and human risks, with contamination of water being a major concern. Decentralized analytical techniques for water monitoring are of high importance. In this work, we present a small, low-cost, and time-effective electrochemical, chip-based microfluidic device for direct electrochemical detection of GLY downstream of a molecularly imprinted polymer (MIP) concentrator. We studied the electrochemical behavior of GLY and its metabolite aminomethylphosphonic acid (AMPA) using cyclic voltammetry with noble metal electrodes in acidic, neutral, and basic media. A chronoamperometric sensor protocol was developed for sensitive and selective GLY measurements on gold electrodes. The optimized protocol was transferred to a chip-based microsensor platform for online and real-time detection of GLY in a microfluidic setup. The results in the range from 0 to 50 μM GLY in 0.5 M H 2 SO 4 show high linearity and a sensitivity of 10.3 ± 0.6 μA mm −2 mM −1 for the chip-based microfluidic platform. Successful recovery of GLY concentrated from untreated tap water and its precise detection from low volumes demonstrates the advantages of our system.