The number of elderly individuals with age-related cognitive decline or dementia is rapidly increasing. Dairy product consumption, including β-lactolin, is beneficial for their cognitive function. The underlying mechanism of β-lactolin’s effects on human brain activity is yet to be investigated. We examined the β-lactolin effects on human cerebral blood flow (CBF) using near-infrared spectroscopy (NIRS) in a placebo-controlled randomized double-blind study, which reported according to the CONSORT guidelines. Fifty healthy participants (aged 45–60 years) were randomly allocated into the β-lactolin or the placebo group (n = 25 each) and supplemented for 6 weeks. During the 6th week, oxy-hemoglobin during the working memory tasks was measured using 34-channels (CHs) NIRS. The changes of oxy-hemoglobin, which represents the CBF, in CH 23 located at the left dorsolateral prefrontal cortex (DLPFC) during the spatial working memory task showed higher statistical significance (false discovery rate (q) = 0.045) in the β-lactolin than in the placebo group. The oxy-Hb changes in CH23 have a co-relationship with the working memory task reaction time. This clinical trial showed an increase in the CBF in the left DLPFC area during the 6-week β-lactolin supplementation. This study contributes to elucidating the underlying mechanisms of β-lactolin on cognitive performance.