One of the major challenges for nanoparticles to be used as a drug/gene delivery platform is their tendency to aggregate in electrolyte solution (physiological environment). The present work introduced the albumin pre-coating strategy that effectively prevented inorganic layered double hydroxide (LDH) nanoparticles with different sizes and interlayer anions from aggregation in phosphate buffer saline and cell culture medium solutions. We found that the key factors influencing the colloidal stability of albumin-coated LDHs included(1) the sequence and speed of reagent addition during the pre-coating process, (2) the albumin/LDH mass ratio, (3) the LDH particle size, and (4) anions intercalated in the LDH. Approximately, LDH nanoparticles with the size of 110 nm were well stabilised at the albumin/LDH mass ratio of 5 : 2 when LDH suspension was added into albumin solution dropwise with vigorous stirring. The albumin pre-coating also enhanced cellular uptake of LDH nanoparticles in Chinese hamster ovary cell culture. The configuration, affinity and adsorption isotherm of albumin on LDH nanoparticles were further investigated. The results in this work imply that the albumin-coating strategy is a potential method to prevent LDH nanoparticle aggregation in in vivo drug/gene delivery.