Providing ancillary services through flexible electric vehicle (EV) charging has the potential to offer extra market benefit for EVs. EV aggregator controlling a fleet of EVs can play a significant role in managing the considerable EV charging demand and bid in the electricity markets. The increasing penetration of EVs has created the feasibility of participating in both the day-ahead energy market and frequency regulation market. This work presents a multi-market optimization model for minimizing the net operation cost of EV charging considering the benefit from performing frequency regulation. A two-level optimization algorithm for EVs controlled by the aggregator is proposed to determine optimal operation strategies of EV aggregations and the charging power of each individual EV. The optimization is able to merge revenue from frequency regulation with the cost reduction objectives of traditional EV charging management. The effectiveness of optimization algorithm is demonstrated by simulating EVs charged at the workplace and residential areas. The increased profitability of participation in the sequential electricity markets has been illustrated. Net operating cost of EV aggregations can be significantly reduced considering both capacity and energy remunerations in the regulation market and the charging demand in the energy market