Background/Introduction: Concurrent electroencephalography and resting-state functional magnetic resonance imaging (rsfMRI) have been widely used for studying the (presumably) awake and alert human brain with high temporal/spatial resolution. Although rsfMRI scans are typically collected while individuals are instructed to focus their eyes on a fixated cross, objective and verified experimental measures to quantify degree of vigilance are not readily available. Electroencephalography (EEG) is the modality extensively used for estimating vigilance, especially during eyes-closed resting state. However, pupil size measured using an eye-tracker device could provide an indirect index of vigilance. Methods: Three 12-min resting scans (eyes open, fixating on the cross) were collected from 10 healthy control participants. We simultaneously collected EEG, fMRI, physiological, and eye-tracker data and investigated the correlation between EEG features, pupil size, and heart rate. Furthermore, we used pupil size and EEG features as regressors to find their correlations with blood-oxygen-level-dependent fMRI measures. Results: EEG frontal and occipital beta power (FOBP) correlates with pupil size changes, an indirect index for locus coeruleus activity implicated in vigilance regulation (r = 0.306, p < 0.001). Moreover, FOBP also correlated with heart rate (r = 0.255, p < 0.001), as well as several brain regions in the anticorrelated network, including the bilateral insula and inferior parietal lobule. Discussion: In this study, we investigated whether simultaneous EEG-fMRI combined with eye-tracker measurements can be used to determine EEG signal feature associated with vigilance measures during eyes-open rsfMRI. Our results support the conclusion that FOBP is an objective measure of vigilance in healthy human subjects.