Lignocellulosic nanofibrils (LCNFs) isolation is recognized as an efficient strategy for maximizing biomass utilization. Nevertheless, achieving a 100% yield presents a formidable challenge. Here, an esterification strategy mediated by the equilibrium moisture in biomass is proposed for LCNFs preparation without the use of catalysts, resulting in a yield exceeding 100%. Different from anhydrous chemical thermomechanical pulp (CTMP0%), the presence of moisture (moisture content of 7 wt%, denoted as CTMP7%) introduces a notably distinct process for the pretreatment of CTMP, comprising the initial disintegration and the post‐esterification steps. The maleic acid, generated through maleic anhydride (MA) hydrolysis, degrades the recalcitrant lignin‐carbohydrate complex (LCC) structures, resulting in esterified CTMP7% (E‐CTMP7%). The highly grafted esters compensate for the mass loss resulting from the partial removal of hydrolyzed lignin and hemicellulose, ensuring a high yield. Following microfluidization, favorable LCNF7% with a high yield (114.4 ± 3.0%) and a high charge content (1.74 ± 0.09 mmol g−1) can be easily produced, surpassing most previous records for LCNFs. Additionally, LCNF7% presented highly processability for filaments, films, and 3D honeycomb structures preparation. These findings provide valuable insights and guidance for achieving a high yield in the isolation of LCNFs from biomass through the mediation of equilibrium moisture.