Bromelain is a major proteolytic enzyme that existed in pineapple core and is widely known for its rich bioactivities, including anti-inflammatory and anti-bacterial. In this research, the isolation, extraction, and purification of bromelain from pineapple core was successfully performed, followed by the formulation of bromelain nanoemulsion, which ended with the in vitro testing on mouse skin to determine its skin permeability. The bromelain activity was also evaluated in this study, whereas the specific activity was determined at 51.36 U/mg and 68.62 U/mg at pineapple core and crude enzyme fractions, respectively. Moreover, the further fractionation using (NH4)2SO4 was performed and resulted in the specific activities of 118.48 U/mg for fraction 0-50% at 12 hr. In addition, three formulas of bromelain nanoemulsion were created in this study and characterized further using organoleptic and in vitro tests. According to the PSA data, the Formula 3 nanoemulsion, which majorly comprised of Tween 80 and small amounts of lecithin, shown a smaller droplet size at 22.04 nm, which corresponds to its higher penetration rate at 587.56 μg/cm2 in 30 minutes. Moreover, the decreased bromelain proteolytic activity in Formula 3 was not significant compared to the other two formulas. Thus, this optimized bromelain nanoemulsion formula can be studied further to examine its anti-bacterial potency through additional in vitro and in vivo tests.