Length of flexor hallucis longus (FHL), localisation of master knot of Henry (MKH) and relationship between MKH and neurovascular bundle are essential for the achievement of FHL tendon transfer. The purpose of this study is to define the localisation of MKH in reference to bony landmarks of the foot, its relationship to plantar neurovascular bundle and to investigate in situ and ex vivo length of FHL tendon in single incision, double incision and minimally invasive techniques. Foot length was examined in 62 feet of 31 soft cadavers (9 males, 22 females). Various parameters including the relationship between MKH and neurovascular bundle, the distances from MKH to medial malleolus (MM), navicular tuberosity (NT) and the first interphalangeal joint of great toe (IP) were measured. Surface localisation of MKH in relation to a line joining the medial end of plantar flexion crease at the base of great toes (MC) to NT (MC-NT line) was determined. Lengths of FHL tendon graft from three surgical techniques were examined. In situ length was measured in the plantar surface of foot and ex vivo length was measured after tendon was cut from its insertion. The mean length of foot was 230.98 ± 15.35 mm with a statistically significant difference between genders in both sides (p < 0.05). No distance was found between medial plantar neurovascular bundle (MPNVB) and MKH. Mean distance of 17.13 ± 3.55 mm was found between lateral plantar neurovascular bundle (LPNVB) and MKH. MKH was located at a mean distance of 117.11 ± 1.00 mm proximal to IP, 26.28 ± 4.75 mm under NT and 59.58 ± 7.51 mm distal to MM with a statistically significant difference of MKH-IP distance between genders in both sides and MKH-NT in right side. MKH was located anterior to NT (66.1%), at NT (27.4%) and posterior to NT (6.5%) on the MC-NT line. Surface localisation of MKH was 94.75 ± 8.43% of MC-NT line from MC with a perpendicular distance of 25.11 ± 5.37 mm below MC-NT line. The in situ and ex vivo tendon lengths from MTJ to ST, to MKH and to IP were 39.05 ± 10.88 mm and 34.43 ± 10.23 mm, 73.45 ± 9.91 mm and 68.63 ± 9.43 mm, 197.98 ± 13.89 and 191.79 ± 14.00 mm, respectively. A statistically significant difference between genders was found in MTJ-IP of in situ and ex vivo length of both sides (p < 0.05). The mean length of tendon between in situ and ex vivo was significantly different in all techniques (p < 0.05). A moderate positive correlation between foot length and tendon length was found in MTJ-IP of both in situ and ex vivo tendon length. A statistically significant difference between in situ and ex vivo tendon length was shown in all harvesting techniques. Surface location of MKH was approximately at 95% of MC-NT line from MC with a perpendicular distance of 25 mm from MC-NT line.