Mental workload (MW) assessment is crucial for understanding human mental state. Cross-subject MW analysis based on electroencephalogram (EEG) signals is an important way. In this paper, a fine-grained and multi-scale motif (FGMSM) features extraction method is proposed, and the proposed features together with original EEG data are used as the input of bidirectional long short-Term memory (Bi-LSTM) to evaluate the cross-subject mental workload. First, the EEG signal of each channel is decomposed based on improved complete ensemble empirical mode decomposition with adaptive noise (ICEEMDAN) algorithm. Second, for the motif structure consisting of three nodes, multi-scale detection is carried out in each intrinsic mode function, and the proportion of each motif structure is extracted as the newly extracted features. Then, the statistical differences of the extracted features between different MW levels are analyzed by using the t-Test, and the features with statistical differences are selected for the cross-subject MW assessment. Finally, based on the public dataset with 26 subjects, Bi-LSTM and a variety of machine learning algorithms are used to classify the levels of cross-subject MW. The results show that the Bi-LSTM classification method with the original EEG data and the proposed features show the most positive results. Therefore, the FGMSM features proposed in this paper with Bi-LSTM provide a new technique for the assessment of cross-subject MW based on EEG signals.