The 35-year-old midfielder will end his 12-year spell at Victoria Park after Pools decided not to take up the option of a further year on his contract.
Humphreys, signed by Chris Turner in 2001, made a total of 542 appearances, scoring 37 goals, for Pools and won the club's player of the year award three times.
Fans' favourite Humphreys, who began his career as a striker at hometown club Sheffield Wednesday, overtook Watty Moore's appearance record for Pools when he made his 473rd outing in a win at Exeter in February 2011.
During the club's centenary season in 2008 he was named Player of the Century in a supporters' poll and completed a successful testimonial season in 2010/11.
Humphreys, who began his playing career at Hillsborough in 1996 and made three appearances for England Under-21s, completed his UEFA A Licence coaching qualification last month.