Nutrient assessment of plants, a key aspect of agricultural crop management and varietal development programs, traditionally is time demanding and labor-intensive. This study proposes a novel methodology to determine leaf nutrient concentrations of citrus trees by using unmanned aerial vehicle (UAV) multispectral imagery and artificial intelligence (AI). The study was conducted in four different citrus field trials, located in Highlands County and in Polk County, Florida, USA. In each location, trials contained either ‘Hamlin’ or ‘Valencia’ sweet orange scion grafted on more than 30 different rootstocks. Leaves were collected and analyzed in the laboratory to determine macro- and micronutrient concentration using traditional chemical methods. Spectral data from tree canopies were obtained in five different bands (red, green, blue, red edge and near-infrared wavelengths) using a UAV equipped with a multispectral camera. The estimation model was developed using a gradient boosting regression tree and evaluated using several metrics including mean absolute percentage error (MAPE), root mean square error, MAPE-coefficient of variance (CV) ratio and difference plot. This novel model determined macronutrients (nitrogen, phosphorus, potassium, magnesium, calcium and sulfur) with high precision (less than 9% and 17% average error for the ‘Hamlin’ and ‘Valencia’ trials, respectively) and micro-nutrients with moderate precision (less than 16% and 30% average error for ‘Hamlin’ and ‘Valencia’ trials, respectively). Overall, this UAV- and AI-based methodology was efficient to determine nutrient concentrations and generate nutrient maps in commercial citrus orchards and could be applied to other crop species.