This studyh focuses on thermal comfort and cost effectiveness of stratum ventilation in subtropical Hong Kong Special Adminstation Region (HKSAR). The need for studying thermal comfort with various air distribution strategies becomes a significant issue recently due to climate change, increasing energy prices and the governmental energy efficiency policy. Stratum ventilation, with air supplied at breathing level, can probably provide satisfactory thermal comfort at a relatively elevated indoor temperature in which less energy use is consumed. It seems that only limited studies on the evaluation of neutral temperature, which is a condition of neither slightly warm nor slightly cool, are supported by actual human comfort surveys. Moreover, study on the related thermal comfort and cost effectiveness as other paradigms in comparison with the mixing and displacement air distribution design is rare. New environmental chamber of laboratory-based air-conditioning systems has been developed for investigating the actual benefit of cost effectiveness and balance of thermal comfort satisfaction with the stratum air distribution strategy under subtropical climates. American Society of Heating, Refrigerating and Air-Conditioning Engineers (ASHRAE) 7-point questionnaires have been collected from human comfort tests so as to estimate the neutral temperature of stratum ventilation in comparison with mixing and displacement ventilation at pre-set conditions. The neutral temperatures of HKSAR people under the mode of mixing, displacement, stratum, modified-stratum-1, modified-stratum-2, and modified-stratum-3 are found to be 24.6℃, 25.1℃, 25.6℃, 26.0℃, 27.1℃ and 27.3℃ at 10 air change per hour (ACH) respectively, which become 24.8℃, 25.3℃, 26.6℃, 27.4℃, and 27.9℃ at 15 ACH respectively. Life cycle assessment results in 10 service year indicate that 7.73% and 7.32% of cost reduction, and 14.52% and 11.91% of greenhouse gas emission reduction in stratum ventilation by comparing with mixing and displacement ventilation. As a result, stratum ventilation should be the best option on both of cost reduction, and less carbon emission in small-to-medium size air-conditioned space for new building and retrofitting existing works.