Walter McCrone was born in Wilmington, Delaware, but he grew up mostly in New York State.  His father was a civil engineer in charge of one of the first DuPont plants to manufacture cellophane.  McCrone received a bachelors degree in chemistry from Cornell University in 1938 and a Ph.D. in organic chemistry from the same institution in 1942.  From 1942 to 1944 he was a post-doctoral researcher at Cornell.  In 1944, McCrone published a detailed study on The Microscopic Examination of High Explosives and Boosters'.
In 1944 McCrone began to work as a microscopist and materials scientist at the Armour Research Foundation, now the Illinois Institute of Technology (IIT) Research Institute.  He was also a professor at IIT and served as assistant chairman of its Chemistry and Chemical Engineering Department.  In 1948, McCrone and IIT electron microscopist Charles F. Tufts organized the first of the meetings that are now the International Microscopy Conference (Inter/Micro).  Among the speakers at the first conference was Nobel laureate Frits Zernike.
In 1956 McCrone left IIT and founded an analytical consulting firm, McCrone Associates, which is now located in Westmont, Illinois.  In 1960, he established the McCrone Research Institute, a nonprofit organization for teaching and research in microscopy and crystallography, based in Chicago.  In 1979 he retired from McCrone Associates in order to dedicate himself to teaching full time. The proceeds from his work as a consulting chemist allowed McCrone to endow the Émile M. Chamot Professorship of Chemistry at Cornell, named in honor of McCrone's university mentor.  According to chemist and forensic scientist John A. Reffner, "during McCrone’s life, he taught microscopy to more students than anyone else in history."
For more than thirty years McCrone edited and published The Microscope, an international quarterly journal of microscopy that had been established in 1937 by the British microscopist Arthur L. E. Barron.  McCrone also wrote more than 400 technical articles along with sixteen books or chapters.  He is credited with expanding the usefulness of the optical microscopy to chemists, who had previously regarded it as primarily a tool for biologists.  One of his publications was the Particle Atlas, first published in 1967, which provided an exhaustive description of small particles and how to identify them with the aid of a microscope.  That work became widely used in forensic laboratories.  The Particle Atlas, which was written in collaboration with other staff members of McCrone Associates, appeared in a six-volume second edition in 1973.  In 1992 it became available in CD-ROM.
Walter McCrone served on the board of directors and as president of the Ada S. McKinley Community Services, a nonprofit social services agency in Chicago.  He died of congestive heart failure at his home in Chicago, at the age of 86.  From 1957 until his death in 2002, he was married to Lucy B. McCrone, née Beman.  The two had met while she was working as an analytical chemist for the management consulting firm Arthur D. Little, in Cambridge, Massachusetts.  After their marriage, Lucy McCrone worked as a chemical microanalyst for McCrone Associates in Chicago and was co-founder and director of the McCrone Research Institute until 1984.