This study deals with the simultaneous determination of sucrose and trehalose in olive leaf samples, with easy sample preparation step, by second derivative UV–Vis spectrophotometry and partial least squares technique. A training set consisting of 31 binary mixture solutions was applied for the construction of PLS model. The proposed procedure was successfully applied for the simultaneous determination of both carbohydrates in laboratory prepared mixtures and in real samples. The real samples were from different growth stages of olive plant. The root mean square error of prediction was determined to be 1.95 and 2.06 for sucrose and trehalose, respectively. Also, limit of detection was 0.21 ppm for sucrose and 0.26 ppm for trehalose. The proposed procedure is rapid, simple, and easy to perform which can be used for routine analysis of both carbohydrates in plant leaves.