Single-cell nanoencapsulation (SCNE) has great potential in the enhancement of therapeutic effects of probiotic microbes. However, the material scope has been limited to water-soluble compounds to avoid non-biocompatible organic solvents that are harmful to living cells. In this work, the SCNE of probiotic Lactobacillus acidophilus with water-insoluble luteolin and Fe3+ ions is achieved by the vortex-assisted, biphasic water-oil system. The process creates L. acidophilus nanoencapsulated in the luteolin-Fe3+ shells that empower the cells with extrinsic properties, such as resistance to lysozyme attack, anti-ROS ability, and α-amylase-inhibition activity, as well as sustaining viability under acidic conditions. The proposed protocol, embracing water-insoluble flavonoids as shell components in SCNE, will be an advanced add-on to the chemical toolbox for the manipulation of living cells at the single-cell level.