Novel in situ chondroitin sulfate (CS) hydrogel was synthesized via phosphine-mediated Michael type addition reaction by mixing precursor solutions of both CS-acrylate and CS-tr0(2-carboxyethyl)phosphine (TCEP) as an electrophile and a nucleophile, respectively. CS-acrylate and CS-TCEP were synthesized in advance by chemical grafting of acrylic acid and TCEP to adipic dihydrazide (ADH)-grafted CS. After verification of chemical grafting of TCEP to CS-ADH by phosphorous peaks in the proton nuclear magnetic resonance spectra (1H NMR) and Fourier transform infrared spectroscopy (FTIR), gel formation was evaluated with a tilting method under different pHs, temperatures and concentrations of the precursor solutions. Precursor solutions spontaneously turned into a gel within a minute to several hours depending on the solution conditions, where a basic pH and higher concentrations and reaction temperatures of the precursor solutions induced quicker gel formation. The fabricated CS hydrogel was thermally stable at low temperatures as observed by both differential scanning calorimeter (DSC) and thermogravimetric analysis (TGA), and also swelled to equilibrium state, leading to 1 to 2 times increase in mass, where a basic medium induced more gel swelling than an acid one. The morphologies of the equilibrated swollen gel showed expansion of its polymer network having pores with a diameter of 13 to 16 μm in dehydrated state. The hydrogel released rhodamine B, a model drug, over approximately 10 h and had 1.08 MPa of compressive modulus for the 10% hydrogel. On the other hand, degradation of the CS hydrogel was controlled by the addition of chondroitinase ABC, along with the reduction of compression strength. The hydrogel had excellent in vitro biocompatibility both inside and on the surface of the CS hydrogel when tested with the live and dead assay of fibroblasts. From these experimental results, we concluded that when TCEP acted as a nucleophilic cross-linking agent of Michael type addition reaction in the synthesis of CS hydrogel, the CS hydrogel demonstrated adequate physicochemical and biological properties.