The presence of multimorbidity in middle-aged and older adults, which reduces their physical activity and quality of life, is a global health challenge. Exercise is one of the most important health behaviors that individuals can engage in. Previous studies have revealed that aerobic exercise training is beneficial for healthy middle-aged and older adults and those with various chronic diseases, but few studies have designed individualized aerobic exercise training for individuals with multimorbidity. Although individuals with multimorbidity are considerably less adherent to physical activity interventions, telephone-based motivational interviewing may help in strengthening motivation and promoting behavioral change for increasing physical activity and health-related physical fitness. This study aimed to examine whether a 12-week individualized aerobic exercise training in a rehabilitation center combined with telephone-based motivational interviewing is effective in promoting physical activity and health-related physical fitness among middle-aged and older adults with multimorbidity. A randomized controlled trial was conducted. Forty-three participants (aged > 40) were recruited and randomly assigned to the intervention group, comparison group, or control group. The participants’ physical activity and health-related physical fitness were assessed at baseline and at 12 weeks. The results indicated that after individualized aerobic exercise training combined with telephone-based motivational interviewing, the participants reported increased total physical activity (Fin = 481.3, p = 0.011), vigorous-intensity physical activity (Fin= 298.9, p = 0.007), dominant and nondominant hand grip (kg) (Fin = 1.96, p = 0.019; Fin = 2.19, p = 0.027, respectively), FEV1/FVC (Fin = 0.045, p = 0.043), VO2 max (ml/kg/min) (Fin = 5.30, p = 0.001), VO2 max predicted (%) (Fin = 21.6, p = 0.001), work (watts) (Fin = 22.5, p = 0.001), and anaerobic threshold (L/min) (Fin = 0.165, p = 0.011). Twelve weeks of individualized aerobic exercise training in the rehabilitation center combined with telephone-based motivational interviewing can increase the total physical activity, vigorous physical activity, and cardiorespiratory fitness of middle-aged and older adults with multimorbidity.