Nowadays, the nutritional value of cookies is relatively low. To reduce wheat flour usage in the cookies, the freeze-dried whole leaf (WL) and chloroplast-rich fraction (CRF) derived from Thai jasmine rice grass were used as a food ingredient to increase the nutrients of cookies. Chloroplast is a rich source of macro and micronutrients. Thus, the use of chloroplast might improve the nutrients in food products. This research aimed to study the physical and chemical characteristics of freeze-dried WL and CRF from Thai jasmine rice grass. The nutritional and physical properties of the butter cookies substituted wheat flour with WL or CRF (0%, 2%, and 4% (w/w)) were also investigated. The result showed that CRF derived from Thai jasmine rice grass contained a significantly larger amount of macro and micronutrients (protein, lipid, ash, -carotene, and total carotenoids) compared with WL (P≤0.05), except for carbohydrate, fiber, total chlorophyll, and total phenolic compound. Furthermore, the cookies replaced wheat flour with 4% WL from Thai jasmine rice grass, containing the highest fiber led to a decreased spread ratio and increased the hardness of cookies. Whilst the cookies substituted with 4% CRF (w/w) were similar in texture to the cookies containing only wheat flour (P>0.05). Moreover, the replacement of wheat flour with freeze-dried WL and CRF derived from Thai jasmine rice grass enhanced nutrients and antioxidant activity compared with cookies in the absence of WL and CRF. The butter cookies with 4% replacement of WL showed the highest level of chlorophyll and total phenolic; the cookies with 4% replacement of CRF contained the largest amount of -carotene (provitamin A) and total carotenoids. However, both cookies containing either WL or CRF were not significantly different in antioxidant activity (P>0.05).