Heating, ventilation and air conditioning systems (HVAC) are the single largest consumer of energy in commercial and residential sectors. Minimizing its energy consumption without compromising indoor air quality (IAQ) and thermal comfort would result in environmental and financial benefits. Currently, most buildings still utilize constant air volume (CAV) systems with on/off control to meet the thermal loads. Such systems, without any consideration of occupancy, may ventilate a zone excessively and result in energy waste. Previous studies showed that CO₂-based demand-controlled ventilation (DCV) methods are the most widely used strategies to determine the optimal level of supply air volume. However, conventional CO₂ mass balanced models do not yield an optimal estimation accuracy. In this study, feed-forward neural network algorithm (FFNN) was proposed to estimate the zone occupancy using CO₂ concentrations, observed occupancy data and the zone schedule. The occupancy prediction result was then utilized to optimize supply fan operation of the air handling unit (AHU) associated with the zone. IAQ and thermal comfort standards were also taken into consideration as the active constraints of this optimization. As for the validation, the experiment was carried out in an auditorium located on a university campus. The results revealed that utilizing neural network occupancy estimation model can reduce the daily ventilation energy by 74.2% when compared to the current on/off control.