This paper reports the development of carbon paste electrode (CPE) modified with biochar (BC) and copper(II) 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31 phtalocyanine (CuHPc) used for the sensitive and selective detection of glyphosate in different matrices. The characterizations of the materials were performed using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and Fourier-transform infrared spectroscopy (FTIR). The analytical efficiency of the modified electrodes (CuHPc/CPE and BC-CuHPc/CPE) used for glyphosate detection was evaluated by square-wave voltammetry (SWV). The application of BC-CuHPc/CPE resulted in an improvement in the electron transfer process on the electrode surface, and this led to an outstanding increase of 100% in its current signal compared to CuHPc/CPE. Under optimized conditions, the BC-CuHPc/CPE sensor presented a linear concentration range of 0.3–4 µmol L−1 and a limit of detection of 0.02 µmol L−1. Interference analysis was conducted using different compounds that can be found in the environment including 2,4-D, atrazine, diuron, urea, and caffeine; the results obtained showed that the compounds did not exert any interference in the current peak of the glyphosate. The proposed method was applied for the determination of glyphosate in river water and food samples, where recovery rates of nearly 100% were obtained. The results demonstrate the accuracy and reliability of the proposed modified electrode. Graphic abstract: [Figure not available: see fulltext.]