An assembly of hybrid hydrogel derived from Carbon Dot (CD), Protoporphyrin IX (PpIX) and DNA is reported here. PpIX and CD were covalently conjugated to 5'-phosphate termini of Cytosine (C) rich single strand DNA (ssDNA) separately. The CD-DNA-PpIX hybrid hydrogel was assembled through the formation of intercalated motif (i-motif) DNA network structure from CD-DNA and PpIX-DNA conjugates where CD act as crosslinkers in the hydrogel as well as energy donor to excite the photosensitizer (PS), PpIX. While hydrogel derived from only PpIX-DNA conjugate showed an inadequate sol-gel transition, the same could be precisely achieved from the CD-DNA-PpIX hybrid hydrogel by controlled pH adjustment. Distinct photophysical properties of CD and PpIX including fluorescence emission potentially enable tracking of the PS loading and hydrogel dissolution that was visually detectable under UV illumination. Reactive Oxygen Species (ROS) generation and subsequent killing of gram-positive bacteria (Staphylococcus aureus (S. aureus)) were observed following excitation of PpIX (acceptor) in the hybrid hydrogel either through energy transfer from CD or by direct irradiation of PpIX with visible light. Complete dissolution of hydro gel and sustained release of PpIX and subsequent ROS generation was achieved over 10-11 days that could kill S. aureus systematically. This study provides a promising strategy to address self-quenching and solubility of PS for its sustained release in Antimicrobial Photodynamic Therapy (A-PDT) applications through smart hydrogel formulation. (C) 2019 Elsevier Inc. All rights reserved.