This paper proposes an iterative negative-unlabeled (NU) learning algorithm for cross-subject detection of passive fatigue from labelled alert (negative) and unlabeled driving EEG data. Approach. Unlike other studies which used manual labeling of the fatigue state, the proposed algorithm (PA) first iteratively uses 29 subjects' alert data and unlabeled driving data to identify the most fatigued block of EEG data in each subject in a cross-subject manner. Subsequently, the PA computes subjects' driving fatigue score. Repeated measures correlations of the score to EEG band powers are then performed. Main results. The PA yields an averaged accuracy of 93.77% ± 8.15% across subjects in detecting fatigue, which is significantly better than the various baselines. The fatigue scores obtained are also significantly positively correlated with theta band power and negatively correlated with beta band power that are known to respectively increase and decrease in presence of passive fatigue. There is a strong negative correlation with alpha band power as well. Significance. The proposed iterative NU learning algorithm is capable of labelling and quantifying the most fatigued block in a cross-subject manner despite the lack of ground truth in the fatigue levels of unlabeled driving EEG data. Together with the significant correlations with theta, alpha and beta band power, the results show promise in the application of the proposed algorithm to detect fatigue from EEG.