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The increased demand for agricultural productivity to support the growing population has resulted in the expanded use of pesticides. However, modern pesticide applications contaminate air, water, soil, and unintentional target species. It is necessary to develop effective and sustainable methods to detect different pesticides within our environment. Surface-enhanced Raman spectroscopy (SERS) has garnered significant attention for its ability to detect and quantify environmental contaminants, as it is a rapid and sensitive technique that requires minimal sample preparation. The present study demonstrates the development of a biowaste-derived nanocellulose-based thin-film that, when integrated with gold nanoparticles, produces a sustainable and reproducible SERS nanosubstrate. In this study, three pesticides (carbaryl, ferbam, and thiabendazole) were sensitively and selectively detected by the combined use of this novel nanocellulose-based SERS nanosubstrate and a portable Raman instrument. The limits of detection were determined to be 1.34, 1.01, and 1.41 mg/L for carbaryl, ferbam, and thiabendazole, respectively, all of which are well below the agricultural application concentrations recommended. SERS signals were collected for both prepared ferbam spray solution and collected sprayed droplets, and it was found that there is no major difference in the signals, indicating that this detection method is reliable to detect pesticide droplets. A commercial pesticide was detectable by the biowaste-derived SERS nanosubstrate. This study is among the first to utilize biowaste-derived nanocellulose to create SERS nanosubstrate for pesticide detection in spray droplets. We demonstrate the high potential of biowaste-derived nanocellulose in combination with the portable Raman technique for agricultural pesticide spray detection.