The work aims to prepare hydrobiobased room temperature phosphorescent ink with bio-based materials as rigid matrix and aromatic sulfonates as luminescent molecules, to explore its performance control factors and anti-counterfeiting application in packaging, to solve the problem that the conventional luminous anti-counterfeiting ink is not environmental friendly, has single anti-counterfeiting performance. The room temperature phosphorescent ink was obtained by dissolving the bio-based materials and aromatic sulfonates in water. The components and contents of bio-based materials and aromatic sulfonates in the ink were modulated, to study their effects on the ink's viscosity and the afterglow time after printing. Through overprinting a series of luminescent inks with different colors and afterglow time, the colorful anti-counterfeiting patterns with dynamic luminescence were achieved to explore its potential in anti-counterfeiting application. The experiment results demonstrated that, in the water-based ink, when the optimal content of bio-based material was 8% and the optimal content of aromatic sulfonates was 0.16%, the afterglow time was the longest after drying. In stenciling printing, when the thickness of stencil was 0.5 mm, optimal printing performance of the anti-counterfeiting pattern could be acquired. In conclusion, bio-based materials with abundant amino and hydroxyl groups (such as carboxymethyl chitosan and sodium alginate) can form rigid structures with strong hydrogen bonding, effectively inhibit the non-radiative transition of aromatic sulfonic sulfonates, improve the afterglow time and luminous efficiency of the prepared ink, and effectively improve the environmental friendliness and anti-counterfeiting performance of anti-counterfeiting ink for packaging.