In order to find an effective method of detecting thrips defect on green-peel citrus, a defect segmentation method was developed using a single threshold value based on combination of characteristic wavelengths principal component analysis (PCA) and B-spline lighting correction method in this study. At first, four characteristic wavelengths (523, 587, 700 and 768 nm) were obtained using PCA of Vis-NIR (visible and near-infrared) bands and analysis of weighting coefficients; secondarily, PCA was performed using characteristic wavelengths and the second principal component (PC2) was selected to classify images; then, B-spline lighting correction method was proposed to overcome the influence of lighting non-uniform on citrus when thrips defect was segmented; finally, thrips defect on citrus was extracted by global threshold segmentation and morphological image processing. The experimental results show that thrips defect in citrus can be detected with an accuracy of 96.5% by characteristic wavelengths PCA and B-spline lighting correction method. This study shows that thrips defect on green-peel citrus can be effectively identified using hyperspectral imaging technology.