OBJECTIVE: Functional near-infrared spectroscopy (fNIRS) was employed to explore the potential relationship between grip strength, 6-minute walking distance (6MWD), and working memory in older adults with mild cognitive impairment (MCI). METHODS: Participants aged 60 years and above were recruited and assessed using the Montreal Cognitive Assessment (MoCA). Grip strength and 6MWD were measured, and fNIRS was utilized to monitor blood flow changes in the prefrontal lobe during an N-back task. RESULTS: A total of 72 participants were recruited, aged between 70 and 95 years, with MoCA scores ranging from 10 to 25 points. The MoCA score was most strongly correlated with 1-back performance (r = -0.423, p < 0.001), followed by 0-back performance, and showed no significant correlation with 2-back performance. The activation levels of several channels in the left dorsolateral prefrontal cortex (ch17, ch19, ch23), right dorsolateral prefrontal cortex (ch8), and right ventrolateral prefrontal cortex (ch2) were significantly higher in the high-performance group for 1-back compared to the low-performance group (p = 0.016, p = 0.008, p = 0.013; p = 0.018; p = 0.043). Grip strength and 6MWD were both significantly correlated with working memory performance under all three levels of cognitive load, with the strongest correlation observed for 1-back performance (grip strength: r = -0.377, p = 0.001; 6MWD: r = -0.382, p = 0.001). Grip strength was positively correlated with the activation levels of ch17 and ch19 (r = 0.234, p = 0.048; r = 0.249, p = 0.035), and significant inter-group differences were found for the activation level of ch19 (p = 0.032). Similarly, 6MWD was positively correlated with the activation levels of ch17 and ch2 (r = 0.312, p = 0.008; r = 0.256, p = 0.030), with significant inter-group differences observed in the activation levels of ch17, ch19, and ch2 (p = 0.007; p = 0.022; p = 0.029). CONCLUSION: Cognitive decline in older adults with MCI was associated with a decline in working memory, with 1-back performance being the most sensitive measure. The brain mechanisms underlying this sensitivity involved higher activation levels in the left dorsolateral prefrontal cortex, right dorsolateral prefrontal cortex, and right ventrolateral prefrontal cortex. Older adults with stronger muscle strength and better cardiopulmonary function maintained better working memory, with higher activation levels in the left dorsolateral prefrontal cortex. However, the specific activation channels were not entirely consistent across individuals. CLINICAL TRIAL NUMBER: Not applicable.