Few studies have been undertaken to develop cognitive functional improvement-focused exercise programs and determine their effect. The objectives of this study were to evaluate the effects of a cognitive enhancement fitness program (CEFP) on short-term memory and serum brain-derived neurotrophic factor (BDNF) levels according to the cognitive state in middle-aged women. A total of 30 healthy volunteers aged 40–59 years were divided into two groups, that is, a mild cognitive impairment (MCI) group and a non-MCI group based on results from the Korean Dementia Screening Questionnaire. A single-session CEFP was conducted over 50 min and consisted of four parts: warm-up, low intensity interval circulation dance exercises, moderate intensity resistance exercises using elastic bands, and cool-down. Serum BDNF levels were measured by ELISA and short-term memory determined by forward digit/word span test was assessed before and after CEFP. After CEFP, forward digit/word span test scores and BDNF levels increased to median 119.2%/115.1% and 118.7%, respectively. After CEFP, the MCI and non-MCI groups produced higher forward digit span test scores (from 6.7 ± 1.5 to 7.5 ± 1.4 points, p = 0.023 and from 6.2 ± 2.0 to 7.0 ± 2.1 points, P=0.011, respectively). After CEFP, forward word span scores and BDNF levels increased (from 3.5 ± 1.7 to 4.6 ± 1.8 points, p = 0.029 and from 610.8 ± 221.1 to 757.9 ± 267.9 pg/ml, p = 0.017, respectively) in non-MCI group only. No group differences were observed between change in short-term memory and change in BDNF. Short-term memory and BDNF levels after CEFP were found to be negatively correlated with age, but pre- to post-intervention changes in short-term memory and BDNF were not. The present study shows that a single, 50-minute CEFP improved short-term memory and increased serum BDNF levels in healthy middle-aged women, especially those without MCI.