This study evaluated Fucoidan from Macrocystis pyrifera as a potential treatment for cervical cancer. The research aimed to examine Fucoidan’s in vivo retention capacities in poloxamer-based in situ gels for vaginal drug delivery systems. Five different thermosensitive gel formulations were developed, each with varying concentrations of Pluronic F127 and F68 polymers. The incorporation of HPMC affected the gelation temperature, viscosity, and bioadhesive strength. The accepted formula, F3, had a bioadhesive value of 5415.93 ± 98.74 dyne/cm2 and could form a gel at physiological temperature. Ex vivo animal models showed that Fucoidan components retained well on vaginal tissue. Only F1, F2, and F3 achieved the media after 8 hours of examination. In vivo evaluation showed F3 had the highest drug concentration retained in the vaginal mucosa of female rats after 8 hours (24,115 ± 4,842 g), slowly removed after 24 hours (13,014 ± 5,596 g). In conclusion, increases in the hydrophilic content of formulations led to the retained hydrogel formula, which increased drug release and lowered intravaginal elimination.