In present work, the heart rate variability (HRV) characteristics, calculated by sample entropy (SampEn), were used to analyze the driving fatigue state at successive driving stages. Combined with the relative power spectrum ratio beta/(theta + alpha), subjective questionnaire, and brain network parameters of electroencephalogram (EEG) signals, the relationships between the different characteristics for driving fatigue were discussed. Thus, it can conclude that the HRV characteristics (RR SampEn and R peaks SampEn), as well as the relative power spectrum ratio beta/(theta + alpha) of the channels (C3, C4, P3, P4), the subjective questionnaire, and the brain network parameters, can effectively detect driving fatigue at various driving stages. In addition, the method for collecting ECG signals from the palm part does not need patch electrodes, is convenient, and will be practical to use in actual driving situations in the future.