Upconversion nanoparticles (UCNPs), typically converting near-infrared (NIR) light into visible luminescence, are promising for bioimaging applications. However, optical multiplexed in vivo upconversion experiments have long been hampered by the exceptional rarity of available luminescence bands in UCNPs that can penetrate deep in tissues. Herein, we describe an approach to accomplish multiplexed upconversion in vivo imaging through time-domain discrimination of tissue-penetrating NIR luminescence at 808 nm (from thulium ions) with a multitude of distinct lifetimes. A tetradomain nanostructure design enables one to regulate energy migration and upconverting processes within confined nanoscopic domains in defined ways, thus yielding high quantum yield upconversion luminescence (maximum ≈ 6.1%, 0.11 W/cm2) with precisely controlled lifetimes that span 2 orders of magnitude (from 78 to 2157 μs). Importantly, intravenous and subcutaneous administration of aqueous form UCNPs into a Kunming mouse demonstrates high-contrast lifetime-colored imaging of them in liver and two abdomen subcutis. Moreover, optical patterns of these UCNPs allow multicolour presentation of a series of deciphered images that are hued with precisely defined lifetimes. The described temporal multiplexed upconversion approach, demonstrated in in vivo imaging and multilevel anticounterfeiting, has implications for high-throughput biosensing, volumetric displays, and diagnosis and therapy.