Nowadays there is a growing trend to find new sources of dietary fibre (DF), such as marine algae by-products that have traditionally been undervalued. In this respect, the aim of the present investigation was firstly to compare two methods of dietary fibre quantification (Englyst and Prosky) and secondly to determine the chemical composition and some of the functional properties of total and insoluble fibres extracted in accordance with the Englyst method. The dietary fibres of dried Ulva lactuca collected from the Tunisian littoral were determined by the Prosky (gravimetric method) and Englyst (enzymatic-chemical method) methods. The two extraction methods (Englyst–Prosky) provided approximately the same values in total fibres (~ 54%). However, they had different insoluble and soluble fibre contents. U. lactuca contained 20.53% and 31.55% of soluble fibres and 34.37% and 21.54% of insoluble fibres using the Prosky and Englyst methods, respectively. The fractionation of the insoluble dietary fibre concentrate revealed that hemicellulose was the most abundant fraction (32.49%), followed by cellulose (16.59%) and “lignin-like” compounds (1.53%). For both fibre concentrates, the main neutral sugar was glucose (20.70%–27.59%), which corresponded to hemicellulose and cellulose. The water holding capacity of insoluble fibre concentrate was relatively high. It varied between 9.32 g and 10.3 g of water/g of dry fibre at 25 °C and 80 °C, respectively. Nevertheless, the oil holding capacity of the insoluble fibre concentrate was not affected by temperature. It was about 1.08 and 1.01 g of oil/g of dry fibre at 25 °C and 80 °C. Despite the significant functional properties of fibre concentrate, the presence of lead limits its use as an ingredient in the food industry.