Identification of target tissues such as tumors is essential to minimize unnecessary resection of normal tissues during surgery. However, conventional tissue markers using metal clips, ink tattooing, and metal-coil embedded pellets are not appropriate for the real-time detection of tumor sites during laparoscopic surgery or long-term follow-up of target tissues without ionizing irradiation. Therefore, the development of safe and novel tissue markers is urgently required for clinical practice. In this study, we developed a near-infrared (NIR) fluorescence marker (MicBall) to achieve precise and accurate marking for laparoscopic surgery, along with long-term marking capability. MicBall generated bright NIR fluorescence comparable to that of an indocyanine green-human serum albumin solution. MicBall is suitable for evading phagocytosis by macrophages, thereby allowing long-term preservation at the injection sites. Furthermore, the introduction of multiple, small-sized, and separate pore structures inside MicBall provided long-term labeling capabilities by preventing unwanted NIR dye release. The morphology, size, and NIR fluorescence of MicBall did not change after γ-ray irradiation for sterilization. Results from in vivo evaluation of MicBall/alginate hydrogels corroborated their potential utility as tissue markers for real-time detection of target sites, as well as for long-term follow-up and subsequent imaging-guided resection of marking sites.