Heparin is still widely used for treatment and prevention of thromboembolic diseases. Due to specific physicochemical properties, it requires frequent parenteral injections. In this study we present the development and in vitro evaluation of an advanced delivery system for prolonged subcutaneous release of heparin. The delivery system consisted of an in situ forming thermoresponsive poloxamer-based platform combined with pH-responsive polyelectrolyte heparin/chitosan nanocomplexes. Thermoresponsive hydrogels were tested for gelation temperature, gel dissolution and in vitro heparin release, whereas polyelectrolyte nanocomplexes were physico-chemically characterized, as well as tested for in vitro cytotoxicity and in vitro heparin release. Hydrogel combined of two poloxamers demonstrated the highest gelation temperature (28.6 °C), while the addition of hydroxypropyl methylcellulose prolonged gel dissolution. On the other hand, nanocomplexes’ dispersions, prepared at 1:1 heparin/chitosan mass ratio and in the concentration range 0.375–1.875 mg/mL, demonstrated mean diameter <400 nm and zeta potential >34 mV. Pharmacokinetics of selected formulations (thermoresponsive hydrogel, nanocomplexes and a dual system consisting of nanocomplexes incorporated into thermoresponsive hydrogel) were studied in rats. Heparin plasma concentration-time profiles revealed a double-peak phenomenon, probably due to heparin diffusion inside the polymer matrix and gel dissolution. Pharmacokinetic parameters were determined by a non-linear mixed effects modeling approach. It was demonstrated that thermoresponsive hydrogel with heparin/chitosan nanocomplexes enabled the lowest absorption rate of heparin into systemic circulation and provided heparin concentration above the prophylaxis threshold for 5 days. In situ gelling thermoresponsive matrix combined with chitosan nanocomplexes present a promising delivery system for heparin, requiring less frequent administration during long-term treatment.