Objective: To explore the association between magnesium levels and the odds of mild cognitive impairment (MCI). Method: In this cross-sectional study of 1006 participants (≥55 years) from China, whole-blood magnesium concentration was measured using inductively coupled plasma mass spectrometry. MCI was diagnosed according to Petersen criteria using self-reported cognitive decline and a neuropsychological test battery, including the trail-making test-part B (TMT-B), auditory verbal learning test (AVLT), digit symbol substitution test (DSST), and verbal fluency test (VFT), which measured the assessment of executive, memory, attention, and language functioning, respectively. A logistic regression was used to assess the relationship between magnesium levels and MCI, and linear regression analyses were performed for the association between magnesium and cognitive function score. Results: The MCI group had a significantly lower concentration of magnesium compared to the Non-MCI group (34.7 ± 9.8 vs. 36.7 ± 9.7,p= 0.017). After adjusting for covariates, a negative association was observed between magnesium levels and MCI. Compared with the lowest quartile (median: 25.4 mg/L), the odds ratio for MCI was 0.53 (95%CI 0.32–0.90) for the highest quartile (median: 48.4 mg/L), and there was an inverse dose–response relationship (pfor trend = 0.009). In addition, higher levels of magnesium were positively correlated with VFT scores (β = 0.37, 95%CI = 0.11–0.62) and DSST scores (β = 0.50, 95%CI = 0.01~0.98) and negatively correlated with TMT scores (β = −1.73, 95%CI = −3.40–−0.07) in the middle-aged and older adults. Conclusions: Whole-blood magnesium was inversely associated with the occurrence of MCI and positively associated with performance in neuropsychological tests assessing attention, executive, and language ability in middle-aged and older adults.