Background: Transcranial photobiomodulation (tPBM), as a novel non-invasive neurostimulation technique, has shown the compelling potential for improving cognitive function in aging population. However, the potential mechanism remains unclear. Neuroimaging studies have found that tPBM-induced physiological changes exist in both targeted and non-targeted brain areas, suggesting the necessity of understanding the modulation mechanism from the perspective of the whole brain level. Objective: This randomized, single-blind, sham-controlled crossover study aimed to investigate the hypothesis that tPBM improved working memory in healthy older adults through the mechanism of optimizing the properties of the resting-state functional brain networks. Methods: A total of 55 right-handed healthy older adults were randomly assigned to sham tPBM session group or active tPBM session group. After a washout interval, they were assigned to the opposite intervention session. Each session included the following: active or sham tPBM application with a 1064-nm laser to the left forehead; before and after, resting-state functional near-infrared spectroscopy (fNIRS) measurements; and the digital nback task. Differences in accuracy and reaction time of the n-back task, and changes in functional connectivity and graph metrics of the brain networks were investigated and compared between the active and sham tPBM sessions. In addition, correlations between tPBM-induced changes in functional brain networks, and the n-back task were examined. Results: The results showed that compared with the sham tPBM session, the accuracy and reaction time during 3back task significantly improved in the active tPBM session. In addition, the global efficiency, local efficiency, nodal efficiency, and functional connectivity significantly increased in the active tPBM session, particularly in the frontoparietal areas. Importantly, the altered 3-back accuracy was positively correlated with the changes of functional connectivity and nodal efficiency mainly in left prefrontal cortex in those who had increased 3-back accuracy in the active tPBM session. Conclusion: This study suggests that tPBM may serve as an effective tool to improve working memory in older adults through the modulation of resting-state functional brain network properties. Investigations in large-scale samples are needed to further validate the findings of this study.