Mah ı, a unique herbal ink used in medieval Assam in India for writing and illustrating manuscripts, is known for its intense colour and durability for centuries, and its non-corrosive nature. Mah ı is distinctly different from the other contemporary inks used elsewhere in the world, including other parts of India. The interesting features of Mah ı are due to its special recipe involving several herbal ingredients in addition to iron sourced from fish blood or the rust of iron tools, with cow urine as an extractant and solvent. The objective of the current study is an in-depth understanding of the traditional method of the preparation of Mah ı, and its characterisation through analysis of the physicochemical properties using spectroscopic and imaging techniques as well as biochemical analysis. A series of model Mah ı samples are prepared following the traditional method with varying ingredients, and their compositions and physicochemical properties are evaluated with respect to the ingredients using standard methods and analytical tools including atomic absorption spectroscopy, tensiometry, ultraviolet ( UV )-visible, Fourier Transform-infrared ( FT-IR), Raman, steady-state fluorescence, UV fluorescence and emission microscopy. The colour of the ink has been attributed to various polyphenols, gallic acid, epigallocatechin gallate, epicatechin, quercetin, kaempferol and tannic acid, and their iron complexes. UV fluorescence and emission microscopy confirmed an autofluorescence indicative of the presence of phenolic and chlorophyll pigments. Solubilisation by glycosidic biosurfactant contributes to stabilisation of Mah ı. The non-corrosive nature of Mah ı has been attributed to its neutral pH and absence of free iron and copper ions.