Wool, which is classed as keratin fibre, is the most important animal fibre in textile industries. Cortical cells of wool, which is the majority component, have been studied in various aspects. However, there are little researches focused on the relationship between wool diameter and cortical cells included ortho-, meso-, and paracortical cells. Moreover, in the past three decades, studies of cortical cells have been restricted to qualitative analysis and the interpretation of a single analytical technique. Thus, this study set out to fill this gap of knowledge. Merino wool fibers with various diameters were analysed by three methodologies based on thermal (differential scanning calorimetry; DSC), optical (light microscopy), and scanning probe microscopy (atomic force microscope; AFM) analysis. Additionally, this study also provided a large scale of quantitative analysis of the relationship between wool diameter and main cortical cells percentages including ortho- and paracortical cells by deconvolution of DSC curve using three Gaussian distributions of equal width. Unlike previous finding, we found that the fine and coarse wools have a different characteristic of denaturation temperature. Only coarser wools (diameter larger than 25 Âµm) show a linear relationship between wool diameters and denaturation temperature. Interestingly, a combined thermal and optical methodological approach showed that the denaturation temperature difference between para- and orthocortical cells drop when wool diameters increase. The decrements in temperature difference between para- and orthocortical cells might then due to a much lower distinguished between ortho- and paracortical cells that observed in the methylene blue staining pattern of coarser wools. However, the relationship between wool diameters and cortical cell percentages is not observed by both thermal and optical analysis. Significantly, the optical analysis confirms the quantitative analysis of cortical cells estimation by DSC deconvolution. Thus, apart from conventional microscopy techniques, DSC deconvolution is a feasible technique to estimate cortical cells percentages. While Janus green staining was used to determine percentage areas of ortho- and paracortical cells, the successful modified methylene blue staining provided the new evident that mesocortical cells might not distinguish enough to classify as a minority of cortical cell types. The AFM image observed also confirm expectations based on earlier staining patterns of Janus green and methylene blue observations. Additionally, all three techniques support that mesocortical cells did not exist. To sum up, the importance of this research is in raising awareness about the issues of employing a multidisciplinary approach to investigate the cellular structural structure and finding the relationship between wool diameter and cortical cells. It is expected that these findings will contribute to a better understanding of the biological and structural basis of wool fibre such as human hair.