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Manganese (Mn) is an essential trace element to humans. However, excessive Mn causes cognitive impairment resulting from injury to the central nervous system within the hippocampus. No ideal biomarker is currently available for evaluating Mn exposure and associated neurotoxicity in the body. Hence, this study used Mn levels in the serum (MnS), teeth (MnT), and hair (MnH) as biomarkers for evaluating the association between Mn exposure and cognitive impairment in Mn-treated rats. A total of 32 male Sprague-Dawley rats were randomly divided into four groups, received 0, 5, 10, and 20 mg/(kg day) of MnCl2·4H2O for 5 days a week for 18 weeks, respectively. Lifetime Mn cumulative dose (LMCD) was used to evaluate external Mn exposure. Hippocampus, serum, teeth, and hair specimens were collected from the rats for Mn determination by graphite furnace atomic absorption spectrometry. Learning and memory functions were assessed using the Morris water maze test. Results showed that chronic Mn exposure increased the hippocampus (MnHip), MnS, MnT, and MnH levels, as well as impaired learning and memory function in rats. MnHip, MnT, and MnH levels were positively correlated with LMCD (r = 0.759, r = 0.925, and r = 0.908, respectively; p 0.05), escape latency (r = 0.862, r = 0.716, and r = 0.814, respectively; p 0.05), and the number of platform crossings (r = -0.734, r = -0.514, and r = -0.566, respectively; p 0.05). No association was observed between MnS levels and the number of platform crossings (r = -0.286, p 0.05). Thus, MnT and MnH detected long-term low-dose Mn exposure. These parameters can be reliable biomarkers for Mn exposure and associated neurotoxicity in Mn-treated rats.